JP6810103B2 - Stem cell aggregate suspension composition, its differentiation method - Google Patents

Description

Federal-sponsored research or developmental statement Some of the research for the completion of the present invention is US Government-funded National Institutes of Health Grant No. It was done using 5R24 RR021313-05. Therefore, the US Government has certain rights to the invention.

Background of the invention Field of invention
The present invention relates to a suspension cell assembly composition that is substantially free of serum and feeder, and a method for differentiating a cell assembly suspension.
To date, no efficient system has provided a large-scale manufacturing process (“scale-up”) for eukaryotic mammalian cells, especially mammalian pluripotency such as human embryonic stem (hES) cells. There was nothing about cells. To maintain hES cells in an undifferentiated state in vitro, hES cells are typically maintained on a mouse fetal fibroblast (MEF) feeder and undergo manual mechanical dissociation (eg, microsurgery). After that, it was passed on to individual colonies. These methods are sufficient for research studies, gene targeting, drug delivery, and in vitro toxicity studies that do not require mass production of undifferentiated or differentiated hES cells. Future clinical applications for stable, large-scale expansion of hES cells, including enzyme passage, require improved methods.

Enzyme expansion of hES cells can be performed, but these methods are technically flawed because hES cells rely on cell-cell interactions as well as para- and autocline signals for survival. Therefore, hES cells prefer the intracellular microenvironment as compared to when they exist as single cells. It has also been reported that enzymatic dissociation of hES cells can lead to abnormal karyotypes, leading to the consequences of genetic and epigenetic changes. The result is a highly supportive culture environment, with the firmness of undifferentiated hES or differentiated hES cells without compromising on long culture periods, pluripotency, pluripotency or genetic stability. It is essential to consider the large-scale expansion (ie, the manufacturing process).

Human pluripotent cells investigate the early stages of human development and offer a rare opportunity for therapeutic involvement in some medical conditions such as diabetes and Parkinson's disease. For example, the use of insulin-producing β-cells obtained from human embryonic stem cells (hESCs) will provide significant improvements to current cell therapy procedures that utilize cells from donor pancreas. Current cell therapies for diabetes that utilize cells from donor pancreas are limited by the lack of high quality islet cells required for transplantation. Cell therapy for Type I diabetes patients require transplantation of pancreatic islet cells of approximately ⁸ × 10 8 (Shapiro et al., 2000, N Engl J Med 343 : 230-238; Shapiro et al., 2001a, Best Pract Res Clin Endocrinol Matab 15: 241-264; Shapiro et al., 2001, British Medical Journal 322: 861) Therefore, at least two healthy donor organs are required to obtain sufficient islet cells for successful organ transplantation.

As a result, embryonic stem (ES) cells represent a powerful model system for the study of pluripotent cell biology and the mechanisms underlying differentiation in early embryos. It also provides opportunities for mammalian genetic manipulation and results in commercial, medical and agricultural uses. In addition, the potential proliferation and differentiation of embryonic stem cells is used to create an unlimited source of cells suitable for migration for the treatment of diseases resulting from cell damage or dysfunction. Early primordial ectoderm (EPL) cells described in international patent application WO99 / 53921, in vivo or in vitro derived ICM / blastomere upper layer, in vivo or in vitro derived primordial ectoderm, primordial germ cells ( Other pluripotent cells and cell lines, including EG cells), malformed cells (EC cells), and pluripotent cells induced by dedifferentiation or nuclear transplantation, have some of these properties and uses. Or will share everything. International patent application WO 97/3203 and US patent number 5,453,357 describe pluripotent cells, including cells from species other than rodents. Human embryonic stem cells are described in International Patent Application WO 00/27995 and US Pat. No. 6,200,806. And human EG cells are described in International Patent Application WO98 / 43679.

The biochemical mechanisms that regulate embryonic stem cell pluripotency and differentiation are very poorly understood. However, the data obtained from the limited experiments available (many anecdotal evidence) show that continuous maintenance of pluripotent embryonic stem cells under in vitro culture conditions is due to the presence of cytokins and growth factors in the extracellular environment. It suggests that it depends on the existence of.

While human ESCs provide a source of starting material for developing significant amounts of high quality differentiated cells for human cell therapy, conditions equivalent to the expected prescriptive guidelines for determining clinical safety and efficacy. These cells must be obtained and / or cultured in. Such guidelines would require the use of chemically defined media. Development of such chemically defined / GMP standard conditions is needed to facilitate the use of cells obtained from hESCs for therapeutic purposes and hESCs in the human body.

In addition, the ultimate use of hESC-based cell replacement therapy will require the development of methods that allow mass culture and differentiation conditions that correspond to regulatory guidelines. Several groups have reported simplified growth conditions for hESCs, but these studies have considerable limitations. However, in general, successful isolation of pluripotent cells, long-term clonal maintenance, genetic manipulation, and germline transmission have been difficult to date.

Most cell culture conditions for stem cells include a serum substitute (KSR) in the medium. (Xu et al., 2005 Stem Cells, 23: 315-323; Xu et al., 2005 Nature Methods, 2: 185-189; Beattier et al., 2005
Stem Cells, 23: 489-495; Amit et al., 2004 Biol. Reprod. , 70: 837-845; James et al., 2005 Development, 132: 1279-1282) KSR is not a highly purified source and contains a crude product of bovine serum albumin (BSA). Others only perform short-term trials, so it is unclear whether those conditions allow the maintenance of pluripotency for long periods of time. (Sato et al., (2004) Nature Med., 10: 55-63; US Patent Publication 2006/0030042 and 2005/02333446). Others showed long-term maintenance of pluripotency in media chemically defined with FGF2, activin A, and insulin, but the cells were plates coated with human serum and cell plating. Raised on something that was "washed out" before. (Valuer et al., 2005 J Cell Sci., 118 (Pt 19): 4495-509) FGF2 is a component of all these media, but it is not clear if it is absolutely necessary; especially some formulations. For objects, it is necessary to use it in high concentrations (up to 100 ng / mL, Xu et al., 2005 Nature Methods, 2: 185-189).

In addition, all of these groups contain insulin in their medium at microg / mL levels or are present by the use of KSR.
Glucose metabolism and "cell survival" through the binding of insulin to insulin receptors
It is usually thought to work in signaling.
At levels above physiological concentrations, however, insulin can bind to the IGFl receptor with low efficiency and confer classical growth factor activity through the PI3 kinase / AKT pathway. The presence / need of insulin at such high levels (microg / mL levels) in KSR or other media manifests major activity through binding to the IGFl receptor expressed by hESCs. It is suggested that this is done (Sperger et al., 2003 PNAS, 100 (23): 13350-13355). The expression of intracellular signaling pathway members in the complete complement of IGFlR and hESCs is likely to imply functional activity of this pathway (Miura et al., 2004 Aging Cell, 3: 333-343). Insulin or IGFl can manifest the major signals required for self-renewal of hESCs. This is suggested by the fact that all conditions developed for culturing hESC include insulin, insulin provided by KSR, or IGFl provided by serum. In support of this concept, cells have been shown to differentiate when PI3 kinase is banned in hESC cultures (D'Amour et al., 2005 Nat. Biotechnol., 23 (12): 1534-41; McLean et al., 2007 Stem Cells 25: 29-38).

Recent publications outline humanized, defined media for hESCs (Ludwig et al., Nature Biotechnology, published).
online January 1,2006, doi: 10.1038 / nbtl 177). However, this recent formulation contains several factors that have been suggested to affect the growth of hESCs, such as FGF2, TGFβ, LiCl, γ-aminobutyric acid and pipecolic acid. It should also be noted that this recently defined cell culture medium contains insulin.

The EGF growth factor family includes at least 14 members, including EGF, TGFβ, heparin-binding EGF (hb-EGF), epiregulin-β (also epiregulin-β (HRG-β), glue cell growth factor, etc. (Called), HRG-α, amphiregulin, betacelulin, and epiregulin, but are not limited to these. All these growth factors contain the EGF domain and are first expressed as a transmembrane protein that produces a soluble ectdomain growth factor that is processed by the metalloproteinase (specifically Adam: ADAM) protein. Alternatively, members of the EGF family interact with homo- or hetero-dimers of ErbBl, 2, 3, and 4 cell surface receptors with different affinities (Jones et al., FEBS Lett, 1999, 447: 227). -231). EGF, TGFα, and hbEGF bind with ErbBl / 1 (EGFR) homodimers with high affinity (range 1-100 nM), while HRG-β has very high affinity with ErbB3 and ErbB4 (range <1 nM). Combine with. The active ErbB receptor signals the PI3 kinase / AKT and MAPK pathways. The most expressed growth factor receptors in hESCs are ErbB2 and ErbB3 (Sperger et al., 2003 PNAS, 100 (23): 133501-13355). HRG-β has previously been shown to support the expansion of mouse primordial germ cells (Toyoda-Ohno et al., 1999 Dev. Biol., 215 (2): 399-406). In addition, overexpression of ErbB2 and subsequent improper activation are associated with oncological development (Neve et al., 2001 Ann. Oncol., 12 Suppl 1: S9-13; Zhou & Hung, 2003 Semin. Oncol. , 30 (5 Suppl 16): 38-48; Yarden, 2001 Oncology, 61 Suppl 2: 1-13). Human ErbB2 (chromosome 17q) and ErbB3 (chromosome 12q) are present on chromosomes observed to accumulate as trisomy of some hESCs (Draper et al., 2004 Nat. Biotechnol., 22 (Draper et al., 2004 Nat. l): 53-4; Cowan et al., 2004 N Engl. J. Med., 350 (13)
: 1353-6; Brimble et al., 2004 Stem Cells Dev. , 13 (6): 585-97; Maitra et al., 2005 Nat. Genet. 37 (10): 1099-103; Mitalipova et al., 2005 Nat. Biotechnol. 23 (1): 19-20; Draper et al., 2004 Stem Cells Dev. , 13 (4): 325-36; Ludwig et al., Nature Biotechnology, public online January 1,2006, doi: 10.1038 / nbtl 177).

ErbB2 and ErbB3 were expressed in mouse blastocysts (Brown et al., 2004 Biol. Report., 71: 2003-2011; Salas-Vidal & Romeli, 2004, Dev Biol., 265: 75-89). However, without being explicitly restricted to the inner cell mass (ICM), ErbBl, EGF, and TGFβ were expressed in human blastocysts (Chia et al., 1995 Development, 1221 (2): 299-307). HB EGF has a growth-promoting effect in human IVF blastocyst cultures (Martin et al., 1998 Hum. Prod., 13 (6): 1645-52; Sargent et al., 1998 Hum. Prod. 13 Suppl 4: 239-48). .. And it has an additional effect on mouse embryonic stem cells in 15% serum (Heo et al., 2005, Am. J. Phy. Cell Physiol., In press). Phenomena before or immediately after transplantation include ErbB2-/-, ErbB3-/-, Neuregurin l-/-(Britsch et al., 1998 Genes Dev., 12: 182-36), ADAM17 /-(Pechon, Et al., 1998 Science, 282: 1281-1284), and ADAM19 − / −, (Horiuchi, 2005 Dev. Biol., 283 (2): 459-71) do not appear to be affected. Therefore, the importance of signaling through the ErbB receptor family of hESCs is previously unclear.

Neuregulin-1 (NRGl) is a large gene that exhibits multiple splicing and protein processing variants. This produces many protein isoforms. (Protein isoforms are collectively referred to herein as neuregulin). Neuregulin is predominantly expressed as a transmembrane protein on the cell surface. The extracellular region contains an immunoglobulin-like domain, a carbohydrate-modifying region, and an EGF domain. NRGl expression isoforms have been previously investigated (Falls, 2003 Exp. Cell Res., 284: 14-30). The cell membrane metalloproteinases ADAM17 and ADAM19 have been shown to treat the transmembrane form of neuregulin-1 into soluble neuregulin / heregulin. HRG-α and -β are cleaved ect domains of neuregulin, including EGF and other domains. Since the EGF domain is responsible for the binding and activation of the ErbB receptor, recombinant molecules containing only this domain can exhibit essentially all of the soluble growth factor effects of this protein (Jones et al., 1999 FEBS). Lett., 447: 227-231). Also, through the interaction of the ErbB receptor with the EGF domain, adjacent cells have a treated transmembrane isoform of neuregulin that appears to trigger juxtacrine signaling.

International patent application WO 99/53021 International patent application WO97 / 32033 U.S. Pat. No. 5,453,357 International patent application WO 00/27995 U.S. Pat. No. 6,200,806 International patent application WO98 / 43679 US Patent Publication 2006/0030042 U.S. Patent Publication 2005/0233446

Shapiro et al., 2000, N Engl J Med 343: 230-238 Shapiro et al., 2001a, Best Pract Res Clin Endocrinol Metab 15: 241-264 Shapiro et al., 2001, British Medical Journal 322: 861 Xu et al., 2005 Stem Cells, 23: 315-323 Xu et al., 2005 Nature Methods, 2: 185-189 Beattier et al., 2005 Stem Cells, 23: 489-495 Amit et al., 2004 Biol. Reprod. , 70: 837-845 James et al., 2005 Development, 132: 1279-128 Sato et al. (2004) Nature Medicine. , 10: 55-632 Valuer et al., 2005 J Cell Sci. , 118 (Pt 19): 4495-509 Sperger et al., 2003 PNAS, 100 (23): 13350-13355 Miura et al., 2004 Aging Cell, 3: 333-343 D'Amour et al., 2005 Nat. Biotechnol. , 23 (12): 1534-41 McLean et al., 2007 Stem Cells 25: 29-38 Ludwig et al., Nature Biotechnology, public online January 1,2006, doi: 10.1038 / nbtl 177 Jones et al., FEBS Lett, 1999, 447: 227-231 Toyoda-Ohno et al., 1999 Dev. Biol. , 215 (2): 399-406 Neve et al., 2001 Ann. Oncol. , 12 Suppl 1: S9-13 Zhou & Hung, 2003 Semin. Oncol. , 30 (5 Suppl 16): 38-48 Yarden, 2001 Oncology, 61 Suppl 2: 1-13 Draper et al., 2004 Nat. Biotechnol. , 22 (l): 53-4 Cowan et al., 2004 N Engl. J. Med. , 350 (13): 1353-6 Brimble et al., 2004 Stem Cells Dev. , 13 (6): 585-97 Maitra et al., 2005 Nat. Genet. 37 (10): 1099-103 Mitalipova et al., 2005 Nat. Biotechnol. 23 (1): 19-20 Draper et al., 2004 Stem Cells Dev. , 13 (4): 325-36 Ludwig et al., Nature Biotechnology, public online January 1,2006, doi: 10.1038 / nbtl 177 Brown et al., 2004 Biol. Reprod. , 71: 2003-2011 Salas-Vidal & Romeli, 2004, Dev Biol. , 265: 75-89 Chia et al., 1995 Development, 1221 (2): 299-307 Martin et al., 1998 Hum. Reprod. , 13 (6): 1645-52 Sargent et al., 1998 Hum. Reprod. 13 Suppl 4: 239-48 Heo et al., 2005, Am. J. Phy. Cell Physiol. ， In press Britsch et al., 1998 Genes Dev. , 12: 1825-36 Peschon, et al., 1998 Science, 282: 1281-1284 Horiuchi, 2005 Dev. Biol. , 283 (2): 459-71 Falls, 2003 Exp. Cell Res. , 284: 14-30 Jones et al., 1999 FEBS Lett. , 447: 227-231

Nevertheless, an important development in the progress of hESC studies to maintain pluripotency in culture is the elucidation of media and cell culture conditions that are compatible with the expected prescriptive guidelines for determining clinical safety and efficacy. Will be. The best results would be the usefulness of chemically defined media for hESCs, but if they are manufactured according to GMP standards, chemically undefined components are acceptable. Thus, methods and compositions for culturing and stabilizing a population of pluripotent stem cells that can be used for therapeutic purposes, wherein the culture composition is defined and / or manufactured in accordance with GMP standards. There is a need for identification. The present invention relates to a composition comprising a basal salt nutrient solution and an ErbB3 ligand, which is essentially serum-free.

The present invention also relates to a composition comprising a basal nutrient solution and means for stimulating ErbB2-derived tyrosine kinase activity in differentiateable cells.
The present invention relates to a method for culturing differentiateable cells. In this method, differentiateable cells are plated on the cell culture surface, and the basal nutrient solution is provided to the differentiateable cells.
A ligand that specifically binds to rbB3 is provided.

The present invention relates to a method for culturing differentiateable cells. In this method, differentiateable cells are plated on the cell culture surface to provide a means for stimulating the differentiateable cells with a basal nutrient solution and ErbB2-derived tyrosine kinase activity in the differentiateable cells.

The present invention is also a method of culturing differentiable cells, in which digestive juice is provided to a layer of differentiable cells contained in a medium chamber prior to digestion, and digestion causes the cell layer to become a single cell. Regarding methods including to. After digestion, single cells are placed in a new tissue culture chamber with a differentiable cell culture medium. There, the differentiateable cell culture medium contains a basal nutrient solution and an ErbB3 ligand. Once cultured, a single differentiateable cell is placed under conditions that allow it to grow and divide.

The present invention relates to a method for generating hES cell aggregates in a suspension from a pluripotent hES adhesion medium. In this method, hES cells are cultured under adhesive growth culture conditions that allow expansion in an undifferentiated state;
The adhered hES cultured cells are separated into a single cell suspension culture (diassociating);
The hES-derived cell aggregate (hES-) in the suspension is stirred by stirring the single cell suspension culture medium until the single cell suspension culture solution forms an hES-derived cell aggregate in the suspension. Contact with first differentiateable culture conditions that allow the formation of derived cell aggregates)
Generates hES-derived cell aggregates in suspension.
In a preferred embodiment, stirring the single cell suspension culture is performed at about 80 rpm to 160 rpm.

The present invention also relates to a method for generating a suspension of hES-derived cell aggregates from a suspension of hES-derived single cells. This method involves culturing hES cells under adhesive growth culture conditions that allow expansion in the undifferentiated state;
Contact undifferentiated hES cells with first differentiation culture conditions suitable for differentiating hES cells to obtain adhered hES-derived cells;
Separation of adhered hES-derived cells into single cell suspension cultures;
By stirring the single cell suspension culture until the single cell suspension culture forms hES-derived cell aggregates in the suspension, the single cell suspension culture solution is added to the suspension. Contact with a second differentiation culture condition that allows the formation of hES-derived cell aggregates to generate hES-derived cell aggregates in suspension. In a preferred embodiment, stirring the single cell suspension culture is performed at about 80 rpm to 160 rpm.

The present invention also optimizes cell concentrations in pluripotent cell cultures, or various growth factors such as FGF10, EGF, KGF, noggin and retinoic acid, apoptosis inhibitors, Rho-kinase inhibitors, And / or how to enrich or alter the composition of the resulting cell culture by varying the concentration of the same, and / or how to alter the population of the hES-derived cell aggregate suspension.

FIG. 1 shows real-time RT PCR of ADAM19, neuregulin 1, and ErbBl-3 in BG01v under specified conditions (8 ng / mL FGF2, 100 ng / mL LR-IGFl, 1 ng / mL Activin A). Expression analysis is shown. GAPDH and OCT4 control reactions are shown. FIG. 2 shows the suppression of proliferation of BG01v cells using AG879. BG01v cells are plated in 6-well trays and then exposed to DMSO (A), 50 nM-20 micro M AGl478 (B), or 100 mM-20 micro M AG879 (C) for 24 hours. It was. After 5 days of culture, the cultures were fixed and stained for alkaline phosphatase activity. AGl478 did not appear to affect proliferation at these concentrations (20 microM shown in B), whereas AG879 substantially slowed cell growth at 5 microm (C). FIG. 3 shows the morphology of BG01v cells cultured in DC-HAIF, a defined culture medium containing 10 ng / mL HRG-β, 10 ng / mL Actibin A, 200 ng / mL LR-IGFl, and 8 ng / mL FGF2. A and B) are shown. Also shown is the morphology (C and D) of BG01v cells cultured in a defined culture medium containing 10 ng / mL HRG-β, 10 ng / mL activin A, and 200 ng / mL LR-IGFl. FIG. 4 shows the expression of ADAM19, neuregulin l, and ErbBl-4 by RT PCR in mouse embryonic stem cells (A) and MEFs (B). FIG. 5 shows suppression of ErbBl and ErbB2 signaling in mouse embryonic stem cells. 2 × ^l0 5 Mouse RlES cells in 10% FBS 1: 1000 in MATRIGEL (TM) were plated on 10% KSR and 1000 U / mL mouse LIF (ESGRO). The next day, DMSO (control carrier), 1-50 microM AG1478, or 1-50 microm AG879 was added with fresh medium. Medium was fixed on day 8 and stained for alkaline phosphatase activity. DMSO (A) and 1-50 micron AGl478 (B and C) did not explicitly suppress proliferation. AG879 may have substantially banned cell growth at 50 microM (comparison of D and F) and slowed growth at 20 microm (E). FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (A) 50 microM AG825 prohibited the growth of BG02 hESCs grown in CM. FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (B) AG825 suppresses ErbB2 Y1248 phosphorylation in hESCs. FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (C) Colony counts for continuous passages of CyT49 hESCs in different combinations of growth factors. FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (D) Cell count analysis of the role of IGFl and HRG in hESC proliferation using BG02 cells (left). FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (E) OCT4 / DAPI immunostaining in repeated experiments showed that IGFl and HRG significantly increased the proportion of OCT4 ⁺ cells compared to ActA / FGF2 conditions. FIG. 6 shows the growth inhibition of BG02 cells grown in a conditioned medium (CM). (F) RTK blotting analysis of BG01 DC-HAIF hESCs pulsed with DC-HAIF for 15 minutes followed by overnight starvation without growth factors. Alternatively, steady-state cultures are shown (left). The average and range of normalized relative intensities were plotted (right). FIG. 7 shows mouse ES cells grown under defined conditions according to a combination of different growth factors. (A) after the 2 × l0 ⁵ cells were grown in a combination of 8 days different growth factors, shows the score of AP ⁺ colonies. (BG) Shows a quadruple image of AP ⁺ colonies grown with a combination of different growth factors. FIG. 8 shows the characterization of human embryonic stem cells maintained in DC-HAIF medium. (A) Analysis of teratomas from BG02 DC-HAIF p25 cells showed pluripotency into ectoderm, mesoderm and endoderm. FIG. 8 shows the characterization of human embryonic stem cells maintained in DC-HAIF medium. (B) Immunostaining of BG02 cells cultured in differentiated 15% FCS / 5% KSR. FIG. 8 shows the characterization of human embryonic stem cells maintained in DC-HAIF medium. (C) High Density Illumina Centrics Humans Containing 47,296 Transcription Probes in BG02 Cells Maintained in CM (64 Passages) or DC-HAIF (10 or 32 Passages in Specified Medium) Venn diagram of transcriptional distribution investigated using 6 expression bead chips (Illumina Sentrix Human-6 Expression Bead chips). FIG. 8 shows the characterization of human embryonic stem cells maintained in DC-HAIF medium. (D) The transcriptional profile of BG02 DC-HAIF p32 cells is very similar to that of BG02 cells maintained in CM (top) and is substantially different from early and terminal subcultures in DC-HAIF. Scatter plot analysis showing that it was not (bottom). FIG. 8 shows the characterization of human embryonic stem cells maintained in DC-HAIF medium. (E) Hierarchical strain formation dendrogram of relative gene representation in different populations made by using Bead studio software. FIG. 9 shows the morphology of cells cultured on humanized extracellular matrix (ECMs) in the presence of DC-HAIF medium. (A) CyT49 cells (diluted 1: 200) grown on MATRIGEL® (diluted 1: 200) with reduced growth factors. CyT49 cells were also grown in tissue culture dishes coated with (B) whole human serum, (C) human fibronectin, and (D) VITROGRO®. FIG. 10 shows the single cell passage of human embryonic stem cells. (A-D) ACCUTASE after passaging and 60mm plating of approximately 5 × 10 ⁵ cells in culture quite common in the (R), BG02 cells image for each stage. (A) Shows that viable cells are attached to the dish 1.5 hours after the initial plate culture. (B) Twenty hours after plate culture, the majority of cells aggregated to form small colonies. These colonies proliferate and expand up to 4 days after plate culture (C), and after 5-6 days, form an epithelial-like monomolecular layer while covering the entire dish (D). (E) Normal male karyotype shown in BG02 cultures passaged 19 times with ACCUTASE® in DC-HAIF. FIG. 11 shows human embryonic stem cells alone using (A) ACCUTASE®, (B) 0.25% Trypsin / EDTA, (C) TrypLE, or (D) Versene. Shows cell morphology after cell passage. FIG. 12 shows the large-scale growth of human embryonic stem cells cultured in DC-HAIF. (A)> 10 Flow cytometric analysis of BG02 cells after expansion into ¹⁰ cells. > 85% of cells expressed OCT4, CD9, SSEA-4, TRA-1-81. (B) RT PCR analysis of the expression of pluripotency markers for OCT4, NANOG, REXl, SOX2, UTFl, CRIPTO, FOXD3, TERT, and DPPA5. No markers of differentiated lineages, α-fetoprotein (AFP), MSXl, and HANDl were detected. (C) Fluorescence in situ hybridization (FISH) using human chromosome-specific repeats showed maintenance of standard copy numbers for hChr12, 17, X and Y. FIG. 13 shows morphology (A) and normal karyotype (B) of hESC BG02 cells grown in defined medium containing HRG-β and IGFl in the absence of FGF2 for a period of 7 generations or longer than 2 months. ) Is shown. FIG. 14 shows a scatter plot analysis of transcription from hESCs (BG02) maintained in DC-HAIF (32s) or DC-HAI (teens). Expression of most transcriptions was detected in both samples. And the transcriptionality was substantially unchanged by culturing hESCs in the absence of exogenous FGF2. Correlation coefficient (R ² ) was determined using all detected transcriptions at expression levels> 0 (all dots) or with transcriptions showing a detection confidence of> 0.99 (). R ² select, indicated by a dotted oval). The diagonal lines indicate the mean and 2-fold difference limits. FIG. 15 shows a hierarchical strain formation dendritic diagram of relative gene expression in different populations of early and late passaged BG02 cells maintained by DC-HAIF. The cells clustered closely (about 0.0075) and BG02 and BG03 cells were similarly maintained in conditioned medium (CM) (about 0.037). Also, BG02 cells maintained in DC-HAI are clustered in close contact with other evaluated hESC populations. As an explanation, in FIG. 15, CM is a conditioned medium. DC is the defined culture medium DC-HAIF, as defined above. ap is ACCUTASE® single cell passage. DC-HAI is the same as DC-HAIF as defined herein, except that it does not contain FGF2. FIG. 16 shows the morphology and alkaline phosphatase staining of BG02 cells cultured in 96-well and 384-well DC-HAIF. One phase contrast imaging of BG02 cells grown in wells ⁽¹⁰⁴ cells / well) (A) and (B) alkaline phosphatase staining of a 96-well plate. One phase contrast imaging of BG02 cells grown in wells (10 ³ cells / well) (C) and (D) alkaline phosphatase staining of 384-well plates. FIG. 17 shows a dark field image of BG02 grown in suspension culture in DC-HAIF. The cultures on the 2nd and 6th days are shown. An image was obtained by using 4 magnifications. FIG. 18 shows the growth rate of DC-HAIF in adhesion and suspension culture. BG02 of 1 × 10 ⁶ cells was plated in parallel wells in adherent and suspension cultures. Cell counts were counted in 1-6 days. FIG. 19 represents qPCR analysis of suspension and adhesive hESCs. BG02 cells (S. hESCs) growing in suspension and those growing in adhesive medium (hESCs) showed comparable levels of lack of OCT4 and SOX17 expression. Adhesive cells differentiated into the final endoderm (DE) and suspended hESCs differentiated into the final endoderm (S.DE d3) in suspension both had the expected significant low expression of OCT4. It showed a high expression of SOXl7. FIG. 20 shows the increase in hESC assembly in the presence of Y27632 in suspension culture. 2 × 10 ⁶ BG02 cells were seeded in 3 mL of DC-HAIF or DC-HAIF + Y27632 and placed in an incubator on a 100 rpm turntable in a 6-well tray. Images of the aggregate were obtained on the 1st and 3rd days. FIG. 21 shows RT PCR analysis of suspension aggregates in the presence of Y27632. RT-PCR was performed on grown cultures to assess the expression of pluripotent markers. Oct4, NANOG, REXl, SOX2, UTFl, CRIPTO, FOXD3 and TERT AND DPPA5 were detected. AFP, MSXl, and HANDl, markers of differentiated linenage, were not detected. FIG. 22A-N shows the marker genes OCT4 (panel A), BRACH (panel B), SOXl7 (panel C), FOXA2 or HNF3beta (panel D), HNFlbeta (panel E), PDXl (panel F) NKX6.1 (panel G). ), NKX2.2 (panel H), INS (panel I), GCG (panel J), SST (panel K), SOX7 (panel L), ZICl (panel M), AFP (panel N), HNF4A (panel O) ), And a bar graph showing the expression mode of PTFlA (panel P). Although this is not a complete list, pluripotent human embryonic stem (hES) cells (stages 0, d0), final endochlorous cells (stage l; d2), PDXl-negative anterior intestinal foliage. Cells (stage 2; d5), PDXl-positive endochlorous cells (stages 3, d8), pancreatic endofollicles (stage 4; d11), pancreatic endocrine precursors, and / or hormone-secreting cells (stage 5; d15) ) Can be used to identify. FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 22 (continued). FIG. 23 is a graph showing the range of cell aggregate diameters (microns) of suspension correlated with the total volume (mL) of medium in culture. FIGS. 24A-D show the hES cell cultures of PDXl (panel A), NKX6.1 (panel B), NGN3 (panel C), and NKX2.2 (panel D) in hES-derived cells. It is a bar graph which shows the expression pattern of a marker gene in correlation with a cell concentration. FIG. 24 (continued). FIG. 24 (continued). FIG. 24 (continued).

Detailed Description of the Invention The methods described herein as opposed to conventionally known methods of regenerative medicine based on seeding individual cells in a polymer scaffold, matrix, and / or gel. Uses a pluripotent hES single cell suspension or a cell aggregate suspension formed from a hES-derived (differentiated) single cell suspension as a constitutive block for tissue formation. Cell aggregates are often composed of hundreds or thousands of individual cells that are linked via adhesive junctions and extracellular matrix, contributing to the final differentiated product as a whole. In this regard, cell aggregates can be defined as the type of tissue that provides many useful properties for more traditionally designed tissues.

In one embodiment of the invention, there is provided a method of producing an hES cell aggregate suspension from a single cell suspension of a pluripotent stem cell culture or hES-derived cell culture. Pluripotent stem cells can initially be cultured on fibroblast feeders, or they can be cultured without feeders. The method of isolating hESC and culturing on human feeder cells are described in the method of culturing human ES cells on human feeder cells (METHODS FOR THE CULTURE OF HUMAN EMBRYONIC STEM CELLS).
It is described in US Pat. No. 7,432,140 under the name ON HUMAN FEEDER CELLS), which is referred to in its entirety as part of this specification. Pluripotent ES cell aggregate suspension cultures made directly from or initiated from hESCs cultured on feeders avoid the need for hESC monolayer formation, such as in adhesive cultures. These methods are described in detail in Examples 17 and 18.

Another embodiment of the invention is a method of producing a cell aggregate suspension directly in a differentiation medium, eg, a differentiation medium reagent capable of activating a TGFβ family or receptor, preferably in a TGFβ family member. provide. Such reagents include, but are not limited to, activin A, activin B, GDF-8, GDF-11, and Nodal. The method of producing a cell aggregate suspension in a differentiation medium is distinguished from other methods and provides the production of a cell aggregate suspension culture in a pluripotent stem cell medium, such as StemPro.

Yet another embodiment of the invention is a differentiated hES cell culture (hES-derived), which is cells from stages 1, 2, 3, 4 as described, for example, in d'Amour 2005 and 2006 above. Provided is a method for producing a cell aggregate suspension formed from "cell culture" or "hES-derived cells"). Thus, the methods for forming cell aggregates described herein are not limited to either hES or hES-derived cell pluripotency or pluripotency stages, but rather usage and cell type optimization. The need for will determine which method is preferred. These methods are described in detail in Examples 19-22.

In another embodiment of the invention, control of the resulting cell composition, eg, control of the proportion of pancreatic endofolliculous cells, pancreatic endocrine cells, and / or foliage cells within PDXl, with different growth factor concentrations. Do it by changing. These methods are described in detail in Example 21.

Unless otherwise stated, the terms used herein should be understood as those of ordinary skill in the art in the art. Also, in addition to the definitions of terms provided below, general definitions in molecular biology can be found in the following literature; Rieger et al., 1991 Glossy of genetics: classical and molecular, 5th Ed. , Berlin: Springer-Verge; and Current Protocols in Molecular Biology, F. et al. M. Ausubel et al., Eds. , Current Protocols, ajoint venture beween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. , (1998 Supplement). When the singular notation is used in the specification and claims, it should be understood to have one or more meanings based on the context in which it is used. For example, "cell" means at least one cell.

Also, in the specification and claims, unless otherwise specified, the amounts of components, proportions or ratios of substances, reaction conditions and other numerical values are understood to be modified by the term "about" in all cases. Will be done. Unless otherwise stated, the numerical parameters described in the specification and claims are approximate values and can vary depending on what is required of the present invention. At the very least, it does not limit the application of the doctrine of equivalents to the claims, and each numerical parameter is reported as at least significant figures, relying on conventional rounding techniques.

The present invention provides a method for the production of hES-derived cell aggregates from hES-derived single cell suspensions. Various mechanical and non-biological elements have been standardized for use in culture-based cell movement and assembly, fluid-mechanical microenvironments that correlate optimal cell assembly growth and properties, and scale-up. As it affects variables, it is necessary to characterize the behavior and effects of cell proliferation and differentiation in various culture vessels, dishes, bioreactors, etc., and various media conditions for the cells, if any. These factors include, but are not limited to, shear rate, shear stress, cell concentration, and various growth factors in the cell medium.

Shear velocity and shear stress are the mechanical properties that define fluid shear in a system. Shear velocity is defined as the velocity of a fluid at a particular distance and is expressed in seconds- ¹ . The shear rate is proportional to the shear stress, and the shear rate (γ) = shear stress (t) / viscosity (micro). Shear stress is defined as a fluid shear force acting in the tangent direction of the cell surface and is expressed as a force per unit area (Dyne / cm ² or N / m ² ). Shear stress is generated by agitating the fluid containing the cells that have been agitated, by agitating the cells through the agitated liquid, or by moving the cells in a dynamic, agitated liquid environment. be able to. The fluid viscosity is typically measured in poise and is 1 poise = 1 dyne second / cm ² = 100 centipoise (cp). The viscosity of water, one of the smallest known viscous fluids, is 0.01 cp. The viscosity of a typical suspension of eukaryotic cells in medium is between 1.0 and 1.1 cp at a temperature of 25 ° C. Both density and temperature affect liquid viscosity.

The fluid velocity also determines whether the flow is laminar or turbulent. Laminar flow occurs when viscous forces are dominant and is characterized by smooth, uniform streamlines at low speeds. In contrast, high velocities and inertial forces predominate in turbulence, characterized by the generation of eddies and vortices, and chaotic flows across space and time. A dimensionless number known as the Reynolds number (Re) is commonly used to quantify the presence of laminar or turbulent flows. The Reynolds number is the ratio of inertia to viscous force and is quantified as (density x velocity x length scale) / (viscosity). Laminar flow is predominant when Re <2300 and turbulent flow is predominant when Re> 4000. Based on this correlation with fluid velocity, the Reynolds number and its result, the degree to which the fluid flow is laminar or turbulent, depends on the shear rate and shear stress experienced by the cells in the suspension. Directly proportional. However, fast shear stress conditions can occur in both laminar and turbulent liquid environments. Initially, the liquid tends to resist movement. For the fluid closest to the gravitational solid surface, a boundary layer or non-flow area occurs immediately adjacent to the surface. This forms a slope of fluid velocity from the surface to the center of the fluid flow. The fluid velocity slope is a function of the fluid velocity and the distance from the boundary layer to the region of the highest fluid velocity. As the liquid flow through or around the container accelerates, the velocity of the flow overcomes the viscosity of the liquid, and the smooth, layered slope is destroyed, forming turbulence. Thomas et al. Showed that putrefaction under turbulent conditions occurs most frequently locally in the region of high shear stress and high energy dissipation rates. See Thomas et al. (1994) Cytotechnology 15: 329-335. These regions occur randomly, but are often found in the boundary layer with the highest velocity gradient. These random fluctuations in fluid velocity can generate regions of very high shear stress, which can have a decisive negative impact on the scale-up of cell culture manufacturing systems. Therefore, in mammalian cell culture production scale-up systems, there is a need for methods that can maintain cell density and viability by controlling the main source of shear in such systems.

Henzaler (Henzaler, 2000, Particle stress in bioreactors, In Advances in Biochemicals)
Engineering / Biotechnology, Shipper, T.K. Ed. Given by Springer-Verlag, Berlin) and Colomer et al. (Colomer, J. et al. 2005. Experimental analysis of coagulation of particles under low-shear flow, Water Res. 39: 2994). The fluid mechanical properties of the bulk fluid in a rotating 6-well dish were calculated by the methods described. The dimensionless stress is equal to the turbulence constant x (aggregate diameter / Kolmogorov microscale) ^ turbulence index. Shear stress is equal to dimensionless stress x fluid density x (kinematic viscosity x force input) ^0.5 . Shear velocity is equal to shear stress / kinematic viscosity. In the force input and Kormogorov microscale calculations, the Reynolds number is required for each rotation speed and is equal to (rotation speed x flask diameter) ^ 2 / viscosity. Since both the force input and the Kormogorov microscale are functions of the Reynolds number, all shear stress and shear velocity calculations vary with rotational speed.

Moreover, shear stress and shear rate are functions of dimensionless stress that depend on the diameter of the aggregate to be formed, so that the shear stress and velocity experienced by the aggregate are expected to increase over time of rotation. .. An example of the calculation is shown in Example 17 for an aggregate diameter of 100-200 microns and a rotation speed between 60-140 rpm. These methods were used to provide an estimate of the average shear of bulk fluid over time. However, the shear stress at the vessel wall is expected to be highest due to the boundary effect. To predict wall shear stress, Lee et al. Proposed that the wall shear stress of a 6-well dish is equal to radius of gyration x (density x kinematic viscosity) (2 x pi x rotational speed) ³ ) ^0.5. Did. Using this approach, wall shear stresses have been calculated for rotational speeds from 60 rpm to 140 rpm and are shown in Example 18. It should be noted that unlike the time-averaged shear stresses experienced by aggregates in bulk fluids, the shear stresses that occur at the walls are independent of the aggregate diameter.

Cultured cell concentration is also an important component of tissue function and is difficult to achieve and / or optimize in two-dimensional traditional tissues (eg, those constructed by adhesion). The effect on cell concentration differentiation will be described in more detail in Example 20. Cell aggregates can overcome this limitation by more accurately assuming a three-dimensional (3D) structure that reflects the cell density and conformation in vivo. As a result, the time it takes for cells to achieve their intended structure can be significantly reduced and / or more consistent and efficient. Moreover, 3D aggregated cells can differentiate and function more optimally. This structure resembles a more normal physiological phenomenon than adhesive culture. Mechanical difficulties in the manufacturing process cause little damage to floating cell aggregates in suspension cultures compared to, for example, mechanical difficulties in adhesive cultures.

Also, typical production scale suspension cultures utilize continuous perfusion of the medium as a method for maintaining cell viability while maximizing cell concentration. In such a relationship, medium exchange gives different effects of fluid shear to adherent cells and suspension aggregates. Immobilized adherent cells are subject to fluid shear stress as the medium flow flows across the cell surface. In contrast, suspended aggregates receive little shear stress across the aggregate surface. This is because the aggregate can freely collapse in response to the applied shearing force. Long-term shear stress is detrimental to adherent embryonic stem cells, and it is expected that the form of suspended aggregates is preferred for optimal survival and function. Efficient manufacturing process for the production of multipotent stem cells derived from multipotent stem cells and / or multipotent progenitor cells, and the above related to shear rate and shear stress. There is a need based on the need for the observed mechanism of. The present invention is a production method for the production of pluripotent stem cells and / or pluripotent progenitor cells obtained for the first time from pluripotent stem cells in suspension form, particularly in cell aggregate suspension form. I will provide a.

As used herein, "single cell suspension" or an equivalent expression thereof, in all mechanical or chemical senses, is hES cell single cell suspension or hES-derived (hES). -derived) means a single cell suspension. There are several methods for dissociating cell clumps to form single cell suspensions from primary tissues, adherent cells and aggregates in culture, eg, physical forces (cells). Mechanical dissociation such as scrapers, narrow-diameter pipettes, fine needle suction, vortex desorption, and grinding by forced filtration through a fine nylon or stainless mesh), enzymes (trypsin, collagenase, Acutase)
(Enzymatic dissociation by such as), or a combination of both. In addition, methods and culture medium conditions that help support the dissociation of hES cells into single cells are useful for defining seeding for expansion, cell selection, multiwell plate assay, culture procedures and cloning. Enables automation of expansion. Thus, one embodiment of the invention is a stable enzymatically dissociated hES of single cells that can support long-term maintenance and efficient expansion of undifferentiated pluripotent or differentiated hES cells. A method for developing a cell or hES-derived cell culture system is provided.

As used herein, the term "contact" (ie, contact of a cell, eg, a differentiateable cell, with a compound) refers to the compound and the cell together in vitro (eg, in culture). Is intended to involve incubating with (adding to cells). The term "contact" refers to in vivo exposure of cells to defined cell media containing ErbB3 ligands and optionally members of the TGF-β family, which may occur naturally in the subject (ie, natural). It is intended not to include exposures that may occur as a result of the physiological process of. The step of cell contact with a defined cell medium containing an ErbB3 ligand and optionally a member of the TGF-β family can be performed in any suitable manner. For example, cells can be treated in adhesive culture or suspension culture. It is understood that cells that have been contacted with the defined medium to stabilize or differentiate the cells can be further processed in a cell differentiation environment.

As used herein, the term "differentiation" refers to the production of a cell type that is a more differentiated type than the cell type from which it is derived. Thus, the term includes partially or finally differentiated cell forms. In general, the differentiated cells obtained from hES cells include hES-derived cells, hES-derived cell aggregate cultures, hES-derived single cell suspensions, or hES-derived cell adhesion cultures, and the like. Say.

As used herein, the term "substantially" refers to a large range or degree, eg, when the term "substantially similar" is used in a method, it is similar in a large range or degree. However, it refers to a method that is different from other methods. As used herein, for example, "substantially free", for example "substantially free of contaminants", "substantially serum-free", "substantially free of insulin or insulin-like growth factors". , Or equivalents thereof, include solutions, media, supplements and excipients in at least 98%, at least 98.5%, at least 99%, or at least 99.5%, or at least 100%. , Serum, contaminants or equivalents. In one embodiment, a defined medium is provided that is serum-free, 100% serum-free, or substantially serum-free. Conversely, if a composition, process, method, solution, medium, supplement, excipient, and the like are described as "substantially similar" or equivalent, then the process, method. , Solutions, etc., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the methods previously described herein, and methods incorporated herein as a whole. It means something similar to.

In certain embodiments of the invention, the term "enriched" means about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the desired cell lineage. , Or a cell culture containing 95% or more.

As used herein, the term "effective amount" or equivalent expression of a compound is capable of differentiating in culture for more than a month in the absence of feeder cells or serum or serum substitutes. A sufficient concentration of compound present in the residual components of a defined medium to act on the stabilization of cells. This amount can be easily determined by those skilled in the art.

As used herein, the term "expression" refers to transcription of a polynucleotide or translation of a polypeptide within a cell, where the level of the molecule is measured in cells that express the molecule rather than in cells that do not. It means being made as high as possible. Methods of measuring molecular expression are well known to those of skill in the art, such as Northern blotting, RT PCR, and in situ.
Hybridization, Western blotting, and immunostaining include, but are not limited to.

As used herein, when referring to cells, cell lines, cell cultures or cell populations, the term "isolated" is substantially isolated from the natural source of cells and cells. , Cell line, cell culture, or cell population is allowed to be cultured in vitro. In addition, the term "isolation" refers to the physical selection of one or more cells from a group of two or more cells, where the cells are selected based on cell morphology and / or expression of various markers. ..

The present invention can be further understood by reference to the following detailed description of preferred embodiments of the present invention and the examples contained therein. However, prior to describing the compositions and methods according to the invention, the invention is not limited to a particular nucleic acid, a particular polypeptide, a particular cell type, a particular host cell, a particular condition, a particular method, or the like. Should be understood. Of course, various improvements and changes are apparent to those skilled in the art.

Standard methods for cloning, DNA isolation, amplification, and purification, standard methods for enzymatic reactions, and various separation techniques, including DNA ligase, DNA polymerase, restriction endonuclease, etc., are known to those skilled in the art. It has been used and is generally adopted. Many standard methods are Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al., 1982 Molecular Cloning, 1982 Molecular Cloning, Col.
Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth. Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; (. Ed) Miller 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular
Biology; Grover Ed. ) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Sellow and Hollywood 1979. 1-4, Plenum Press, New York; The abbreviations and terminology used are considered standard in this field and are commonly used in specialized journals such as those cited herein.

The present invention relates to a composition and method comprising a basal nutrient solution, an effective amount of ErbB3 ligand, and the composition of the present invention is substantially serum-free. The compositions and methods of the present invention are useful for culturing cells, especially differentiateable cells. It is understood that different components can be added to the cell culture at different points during the culture of the differentiateable cells. The medium can contain components other than those described herein. However, at one point during media preparation, or during culture of differentiateable cells, the defined medium is contemplated to include a basal nutrient solution and means of activating ErbB2-derived tyrosine kinase.

The basal nutrient solutions described herein are used to maintain cell growth and viability of hES, but in other embodiments of the invention, pluripotent or pluripotent cell differentiation. To maintain, alternative stem cell cultures work substantially similarly, such as KSR (Invitrogen), foreign-free KSR (xeno-free KSR) (Invitrogen), StemPro® (Invitrogen), mTeSR. (Registered Trademarks) l (Stem Cell Technologies), HESCGRO (Millipore), DMEM-based medium, etc., but are not limited thereto.

In another embodiment, hES cells are cultured in the presence and / or absence of extracellular matrix protein (ECM), such as MATRIGEL, in the defined medium described herein. Human embryonic stem cells cultured in the absence of ECM contain approximately 0.5-10% human serum (hS) or hS enriched from 300K and / or 100K cutoff spin columns (Microcon). Including minutes. A suspension of hES cell aggregates can be prepared by directly incubating hES cells in a medium containing the hS or hS concentrated fraction directly. After incubating the hS or hS concentrated fraction and medium vessel for about 30 minutes, 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 12 hours, and 24 hours at 37 ° C., hES cells. An aggregate suspension can be produced. Plating efficiency for hES cells in hS or hS concentrated fraction-containing medium is described in PCT / US2007 / 062755 for hES cells cultured in DC-HAIF, or DC-using MATRIGEL as ECM. It was comparable to that observed in hES cells cultured in HAIF medium, or hES cells cultured in other similar medium. Culturing hES cells in a defined medium that is substantially serum-free is a pluripotent stem cell medium containing human stems of US Application No. 11/8875, 057, filed October 19, 2007. Methods and compositions for Fiders (METHODS AND COMPOSITIONS FOR FEEDER PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM), which are incorporated herein by reference.

In a different embodiment, the hES cell aggregate suspension was cultured in a substantially serum-free medium in the absence of exogenously added fibroblast growth factor (FGF). This is a serum-free medium from Thomson et al., US Pat. No. 7,005,252, but requires culturing hES cells in a medium containing FGF and other extrinsically added growth factors. It is distinguished from the invention to be used.

Cellular control can act through transduction of extracellular signals across the membrane, leading to regulation of biochemical pathways within the cell. Protein phosphorylation represents a process in which intracellular signals are propagated from molecule to molecule, ultimately resulting in a cellular response. These signaling cascades are highly regulated and often overlap, as evidenced by the presence of phosphatases and many protein kinases. Protein tyrosine kinases are known to play an important role in the development of many pathologies, including diabetes and carcinoma, in humans and have been reported to be involved in a variety of congenital syndromes. Serine threonine kinase (eg, Rho-kinase) is a class of enzymes that, if inhibited, treats diabetes, cancer, various inflammatory cardiovascular diseases, and other human diseases, including AIDS. Can be relevant to. Most of the inhibitors identified or designed so far act at the ATP binding site. Such ATP competitive inhibitors showed the ability to target more poorly conserved regions of the ATP binding site.

The Rho-kinase family of small GTP-binding proteins contains at least 10 members, including Rho AE and G, Rac1 and 2, Cdc42, and TC10. Inhibitors are often referred to as ROK or ROCK inhibitors, and they are used interchangeably herein. The effector domains of RhoA, RhoB, and RhoC appear to have the same amino acid sequence and similar intracellular targets. Rho-kinase operates as the primary downstream mediator of Rho and
It exists as two isoforms α (ROCK2) and β (ROCKl). Rho-kinase family proteins have a catalytic (kinase) domain in the N-terminal domain, a coiled coil region in the middle, and putative plextrin homology (putative) in the C-terminal region.
There is a pleckstrin-homology (PH) domain. The Rho-binding domain of ROCK is localized to the C-terminal portion of the coiled coil region, and the GTP binding form of Rho results in enhanced kinase activity. Information initiated by many agonists of the Rho / Rho-kinase mediator pathway, including angiotensin II, serotonin, thrombin, endoserin-1, norepinephrine, platelet-derived growth factor, ATP / ADP, and extracellular nucleotides, and urotensin II. Plays an important role in communication. In the regulation of target agonists / substrates, Rho-kinases play important roles in a variety of cellular functions, including smooth muscle contraction, actin cytoskeletal tissue, cell adhesion, automotility, and gene expression. Also, due to the role of the Rho-kinase protein in mediating many cell functions that are perceived to be associated with the etiology of arteriosclerosis, inhibitors of this kinase appear to promote atherosclerosis. It may be useful in the treatment or prevention of various atherosclerotic cardiovascular diseases associated with endothelium contraction and increased endothelium permeability. Thus, in other embodiments of the invention, agents that promote and / or support cell survival include various cell culture media, such as the Rho-kinase inhibitors Y-27632, Insulin, and H-1152P. It is added to ITS (insulin / iron-binding globulin / selenium; Gibco). These cell survival promoters are partially isolated, such as foregut endoderm, pancreatic endoderm, pancreatic epithelium, pancreatic stem cell population, and similar, particularly isolated pancreatic endoderm and pancreatic stem cell population. It functions by promoting reassociation of the hES cells or cultures derived from the hES. Increased survival of hES or hES-derived cells, with or without extracellular matrix, regardless of whether the cells were produced in suspended cell aggregates or from adhesive plate cultures, Achieved with or without serum and with or without feeder. Increased survival of these cell populations facilitates and improves purification systems using cell sorters. Therefore, improved cell recovery. The use of Rho-kinase inhibitors such as Y27632 allows expansion of hES-derived cell types by promoting survival from single cells isolated by continuous passage or cryopreservation. Rho-kinase inhibitors such as Y27632 have been tested in hES and hES-derived cell cultures, and Rho-kinase inhibitors can be applied to other cell forms, eg, generally epithelial cells such as lung, thoracic gland, kidney, neurogenic. It can be applied to cell types such as retinal pigment epithelial cells.

As used herein, the term "differentiable cell" refers to a cell or cell assembly that can differentiate into at least partially mature cells, or cell differentiation, such as fusion with other cells, or at least a portion. Used to describe those capable of participating in differentiation into mature cells. As used herein, "partially mature cells", "progenitor cells", "immature cells", "precusor cells". ) ”,“ Multipotent cells ”, and equivalent terms include, for example, final endochlorous cells, PDXl-negative progenitor endometrial cells, PDXl-positive prepancreatic endofollicles. Includes progenitor differentiated cells such as PDXl-positive pancreatic endofollicles, PDXl-positive pancreatic endoblast edge cells. All exhibit at least one characteristic of a mature cell from the same organ or tissue, eg, a genetic phenotype such as morphology or protein expression, but can further differentiate into at least one other cell type. For example, normal, mature hepatocytes normally express proteins such as albumin, fibringen, α-1-antitrypsin, prothrombin coagulation factor, iron-binding globulin, and detoxifying enzymes such as cytochrome P-450. Thus, in the present invention, "partially mature hepatocytes" can express albumin or another protein, or begin to have the appearance or function of normal, mature hepatocytes. it can.

In contrast to previously known cell aggregates that may differ in both size and shape, the cell aggregates and methods described herein have a narrow size and shape distribution. Have. That is, cell aggregates are substantially uniform in size and / or shape. Homogeneity in cell assembly size is important for differentiation performance and culture homogeneity. Assuming equal permeability, the diffusion of oxygen and nutrients into the center of large aggregates is slower when basic mass transport analysis is applied to aggregates, compared to diffusion into smaller aggregates. It is expected to be. Cultures with a mixture of aggregates of different diameters are uniform (size shape) cells because the differentiation of aggregated embryonic stem cells into pancreatic linenage cells depends on the temporary application of specific growth factors. Compared to aggregates, they tend to be out-of-sync cultures. This mixture of cell aggregates results in heterogeneity and poor differentiation performance, eventually failing to adapt itself to production, scale-up, and production. The cell aggregates used herein can be of various shapes, such as spheres, cylinders (preferably having equal height and diameter), or rods. Although aggregates of other shapes can be used in one embodiment of the invention, it is generally desirable that the cell aggregates be spherical or tubular. In another embodiment, the cell aggregate is a sphere and is substantially constant in size and shape. For example, if cell aggregates differ in size or are not uniform, it will be difficult to reliably manufacture and carry out large-scale cell scale-up processes. Thus, as used herein, the phrase "substantially constant" or "substantially constant in size and shape" or its equivalents refer to the spread in the uniformity of the aggregate. , It is less than about 20%. In another embodiment, the spread in uniformity of the aggregate is about 15% or less, 10% or less, or 5% or less.

The exact number of cells per set is not important, but the size of each set (and thus the number of cells per set) is limited by the ability of oxygen and nutrients to diffuse into the central cells, and this number. Will be found by those skilled in the art to vary depending on the cell type and the nutritional demand for this cell type. The cell assembly can contain the smallest number of cells per set (eg, 2 or 3 cells), or can contain hundreds or thousands of cells per set. A cell assembly usually contains hundreds to thousands of cells per assembly. For the purposes of the present invention, cell aggregates typically have a size of about 50 to about 600 microns, but vary depending on the cell type, and the size can be even smaller or larger than within this range. it can. In one embodiment, the size of the cell aggregate is from about 50 microns to about 250 microns, or about 75 to 200 microns, preferably about 100 to 150 microns. In contrast, in aggregates of tubular or non-spherical cells that can be obtained in suspension, the diameters based on minor and major axes (eg, X, Y, and Z axes) are not equal. .. These non-spherical cell aggregates tend to be large in size and have a diameter and height of about 500 to 600 microns. However, if they were not differentiated, these non-spherical hES cell aggregates become spheres once differentiation is initiated by the method described herein. Non-spherical cell populations include, but are not limited to, tubular and cubic cell populations, but are still constant in size and shape.

Many cell types can be used to form the cell aggregates described herein. In general, the choice of cell type will vary depending on the three-dimensional structure designed (eg, pancreas, liver, lungs, kidneys, heart, bladder, blood vessels, and various organ structures including the like). For example
If the three-dimensional structure is the pancreas, the cell aggregate is one or more cell types commonly found in the pancreas (eg, endocrine cells such as insulin, glucagon, ghrelin, somatostatin type cells, endothelial cells, smooth muscle cells, etc.). Would include in an advantageous way. One of ordinary skill in the art can choose the appropriate cell shape for the cell aggregate as desired based on the type of steric tissue or organ. Non-limiting examples of suitable cell types include cells (eg, adult and embryonic), contractile or muscle cells (eg, epithelial and smooth muscle cells), nervous system cells (eg, glue). Cells, dendritic, and neurons), connective tissues (cells that differentiate into bone, cartilage, osteogenic and chondrogenic cells, and lymphoid tissues), parenchymal cells, epithelial cells (lining in cavities, vessels or channels) Endeavor cells), exocrine epithelial cells, epithelial absorptive cells, keratinized epithelial cells (eg, keratinocytes and corneal epithelial cells), extracellular matrix secretory cells, mucosal epithelial cells, kidney epithelial cells, lung epithelial cells, breast epithelium Examples include cells, and similar, and undifferentiated cells (eg, embryonic cells, stem cells, other progenitor cells, etc.).

The cell assembly described herein may be a homo cell assembly or a hetero cell assembly. As used herein, a cell assembly of "homo cells", "single cells" or an equivalent representation thereof refers to multiple cell aggregates in suspension. Here, each cell assembly contains a plurality of living cells that are substantially single cells. For example, the hES cell assembly produced by the methods herein can be substantially homozygous, can substantially consist of pluripotent hES cells, and substantially from the final endochlorous cells. Can be substantially composed of anterior intestinal foliage cells, can be substantially composed of pancreatic intrapancreatic foliage cells, and further PDXl-positive prepancreatic endoplasmic foliage cells, PDXl- It can be positive pancreatic endochlorous cells, PDXl-positive pancreatic endoblasts, pancreatic endocrine precursor cells, pancreatic endocrine cells, and the like.

As used herein, the term "substantially" or "essentially" means that "de minimus" or small amounts of components or cells are present within the cell aggregate suspension type. For example, the cell aggregates in the suspensions described herein are "essentially or substantially homogeneous", "essentially or substantially homozygous", and " "Essentially hES cells", "essentially or substantially final endochlorous cells", "essentially or substantially anterior intestinal foliage cells", "essentially Or "substantially PDXl-negative anterior intestinal foliage cells", "essentially or substantially PDXl-positive prepancreatic intrapyramidal foliage cells", "essentially or substantially PDXl- "Positive pancreatic endoblasts or precursor cells", "essentially or substantially PDXl-positive pancreatic endoblast edge cells", "essentially or substantially pancreatic endocrine progenitor cells" , "Cells of the pancreatic endocrine gland essentially or substantially", and the like.

Substantially homo-cell aggregate suspension cultures are, for example, less than about 50% hESCs, less than about 45% hESCs, less than about 40% hESCs, less than about 35% hESCs, less than about 30%. HESCs, less than about 25% hESCs, less than about 20% hESCs, less than about 15% hESCs, less than about 10% hESCs, less than about 5% hESCs, less than about 4% hESCs, less than about 3% A hES-derived cell aggregate suspension culture comprising hESCs, less than about 2% hESCs or less than about 1% hESCs, based on the total number of hES-derived cells in the culture. In other words, hES-derived cell aggregate suspension cultures, such as PDXl-negative progenitor endoderm, PDX-positive prepancreatic endoderm cells, PDXl-positive pancreatic endoderm or progenitor cells. , PDXl-positive pancreatic end cells, pancreatic endocrine progenitor cells, and pancreatic endocrine cells at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , At least 90%, or at least 95%.

As used herein, "hetero-cellular" and "multi-cellular" or equivalent expressions are cell aggregates, the respective cell aggregates. Is a cell assembly containing multiple cells of at least 2, 3, 4, 5, 6 or more cell types, or at least one cell type and non-cellular component, such as an extracellular matrix (ECM) substance (eg, collagen, fibronectin). , Laminin, elastin, and / or proteoglycan). It is known in the art that such ECM components can be naturally secreted by cells, or that cells alter expression levels of ECM substances and / or cell adhesion molecules such as lectins, integrins, immunoglobulins, or cadherins. It can be genetically engineered in any suitable manner that has been used. In another embodiment, either the natural ECM material or any synthetic material that mimics the ECM material can be incorporated into the aggregate during assembly formation. The production methods described herein for hES-derived cell aggregates, such as, for example, pancreatic epithelium or pancreatic endoblast cell aggregates (or stage 4 cell aggregates), are substantially from pancreatic epithelium or endoblast cells. However, it can also consist of other nonpancreatic epithelial type cells with a small cell count, or endometrial precursor cells, and pancreatic endocrine cells (eg, insulin secretory cells).

Homo- or hetero-cell aggregates produced by the suspension methods described herein are cells called embryonic bodies (EBs), unlike the cell aggregates described in the art. Not the same as an aggregate. EBs are maintained when ES cells are overgrown in a monolayer culture, or are maintained in a suspension culture within an undefined medium, or are non-derived protocols towards multiple germ layer tissues (ie, random). The embryonic body is clearly distinguished from the cell assembly described herein because it is a cell assembly of differentiated and undifferentiated cells that appears when differentiated through differentiation. In contrast, the invention discussed in detail in Examples 17 and 20 enzymatically separates hES cells in adhesive plate cultures to form single cell suspensions, which in turn combine multiple cells. Cell aggregates are formed and these cell aggregate suspension cultures are used for differentiation, as substantially described in d'Amour 2005 and 2006 above. The other differences between EBs and the cell assembly of the present invention are further discussed below.

In addition, other methods of forming embryoid bodies (EBs) will be described. As used herein, the terms "embryoid bodies", "aggregate bodies" or equivalents are used when ES cells are overgrown in a monolayer culture. Differentiated and undifferentiated that appear when maintained in suspension culture within an undefined medium or differentiated via a non-derived protocol (ie, random differentiation) towards multiple embryonic tissues. Refers to a cell aggregate of cells. That is, EBs are not formed from a single cell suspension of pluripotent stem cells as described herein. Also, EBs are not formed from adherent cultures of hES-derived pluripotent cells. These characteristics alone also make a clear distinction between the embryoid body and the present invention.

The embryoid body is usually a mixture of different cell types, which are several germ layers, which can be identified by morphological criteria. An embryoid body is from embryonic stem (ES) cells, most of which are non-derived differentiated, i.e., for example, undifferentiated cells exposed to high concentration of serum in the absence of defined growth factors. Morphological structures composed of the resulting population of cells are usually referred to. Under culture conditions suitable for EB formation (eg, removal of leukemia suppressors for mouse embryonic stem cells, or other similar blocking factors), embryonic stem cells proliferate and form small mass cells. And begin to differentiate. First, in response to about 1-4 days of differentiation for human embryonic stem cells, cell clumps form a layer of endoderm cells on top of the outer layer. It is considered to be a "simple embryoid body". Second, "complex embryoid bodies" are formed in response to approximately 3-20 days of post-differentiation for human embryonic stem cells. It is characterized by large-scale differentiation of ectodermal and mesoderm cells and derivative tissues. As used herein, EBs include both simple and complex EBs, unless considered to vary by context. Decisions regarding when embryoid bodies are formed by culturing ES cells are routinely made by one of ordinary skill in the art, for example, by visual inspection of morphology. Depending on the culture conditions, floating masses of about 20 or more cells are considered to be EBs. For example, Schmitt et al. (1991) Genes Dev. 5,728-740; Doetschman et al. (1985) J. Mol. Embryol. Exp. Morph. See 87, 27-45. The term also refers to a structure equivalent to that obtained from primordial germ cells, which are early cells extracted from the embryonic gonad region. For example, Shamblott, et al. (1998) Proc. Natl. Acad. Sci. See USA 95,13726. Also, primordial germ cells, sometimes referred to in the art as EG cells or germ cells, can form pluripotent embryonic stem cells capable of inducing embryoid bodies when treated with appropriate factor groups. See, for example, US Pat. No. 5,670,372, and Shamblott et al., Supra.

There are various methods for making EBs, such as (2008) Nature Protocol 3 (5): spin embryoid bodies described in 468-776, Bauwens et al. (2008) above. EBs are formed from single cell suspensions plated on micropatterned extracellular matrix islands as described. However, these methods are not cost-effective and not very efficient for large-scale production (manufacturing) of hES cells and cells derived by hES. This is because too many steps are required before scale-up production actually begins. For example, in Bauwens et al., HES cells must first be seeded with reduced growth factor MATRIGEL® before the cells are selected to initiate suspension culture. The time and cost of this method is cumbersome as it requires a custom designed micro-patterned tissue culture plate. Moreover, the method of Ng et al. Is not cost efficient, as large-scale production of hES cells and cells derived from hES-requires the use of a centrifuge to produce a more uniform EB. Finally, in all these methodologies, cell aggregates are not made from single cell suspensions of pluripotent stem cells, as in the present invention.

The embryo-like body is different from the cell assembly described in the present invention, and is a cell assembly made up of a large number of cell types from three germ layers, and typically an aggregate of undifferentiated embryonic stem cells. A cell aggregate produced by exposure to non-directed differentiation signals, such as 20% bovine embryonic serum. The result of this non-tropic methodology is a mixture of cell types intended to mimic normal fetal growth in vitro. This approach is useful for examining fetal growth at the basic research level, but it is primarily concerned with cell yield, population identity, population purity, batch consistency, safety, cell function, and commodity cost. However, it cannot be applied to all large-scale cell therapy manufacturing processes. Moreover, despite any enrichment strategy used to purify a given cell type from the embryoid body, the differentiation protocol does not provide a direct approach to developing a large population of single cell types. Following that, pollutant populations will always be dominant and will prevent any attempt to purify a particular population. All previous studies on creating and differentiating embryonic stem cell aggregates have one or more of the following components in their methodology:
1) Use of mice instead of human embryonic stem cells 2) Forced assembly protocol that relies on efferent precipitation to aggregate cells rather than the normal cell adhesion process 3) Assembly of cell clusters under static conditions 4) Non-single Formation of aggregates by cell dissociation or scraping of surface cells 5) Indirect differentiation of cell aggregates using 15-20% fetal bovine serum, resulting in the formation of cell types of all embryonic bodies and germ layers ..
To our knowledge, the only study that does not utilize 15-20% FCS to differentiate embryoid bodies is that cell aggregates are formed in forced aggregates, then the aggregates are immediately mesoderm. A protocol for differentiating germ layers using a suitable medium is described (Ng et al., Blood. 2005 106 (5): 1601). However, this work is irrelevant to the present invention as the researchers transfer the embryoid bodies to non-aggregated adherent cultures after static assembly for 10-12 days. In contrast to conventional work, the present invention 1) isolates human embryonic stem cells into single cells and rotationally cultures them at an optimized shear rate to improve control of assembly diameter and cell survival. Formation of aggregates by
2) Next, directly differentiate the ES cell aggregate into the final endoderm, differentiate into the anterior endoderm, differentiate into the prepancreatic anterior endoderm, then into the pancreatic endoderm and finally into the pancreatic endocrine cells. Differentiate.
This differentiation protocol is a highly efficient and minimally contaminated population that produces the final endoderm and pancreatic linenage population. Moreover, in contrast to all other published studies, this approach to embryonic stem cell assembly and differentiation does not create embryoid bodies.

In contrast to embryoid bodies that are a mixture of differentiated and undifferentiated cells, typically consisting of cells from several germ layers and undergoing random differentiation, the cell aggregates described herein are: It is essentially or substantially homo-cell and exists as a population of pluripotent, bipotent, or unipotent type cells, such as embryonic cells, final endoderm, preintestinal endoderm. , PDXl-positive pancreatic endoderm, pancreatic endocrine cells, and the like.

The present invention provides an economically efficient manufacturing process or method capable of producing cell aggregates of substantially constant size and shape using a process that is reproducible and easily applicable to large scale manufacturing. , Solve the above problems. In one particular embodiment, the differentiateable cells are expanded in suspension culture using the cell medium of the invention. In another particular embodiment, differentiateable cells are retained and expanded in suspension. That is, they remain undifferentiated or are prevented from further differentiating. When the term "expand" is used in the art in connection with cell proliferation, it refers to cell proliferation and increased cell number, preferably viable cell number. In a specific embodiment, the cells are expanded by culturing in a culture suspension for about 1 day, i.e. about 24 hours or more. In a more specific embodiment, the cells are cultured in suspension culture for at least 1, 2, 3, 4, 5, 6, 7 days, or at least 2, 3, 4, 5, 6, 7, 8 weeks. Expand by.

The differentiation culture conditions and hES-derived cell types described herein are substantially similar to those described in d'Amour et al., 2006, supra. d'Amour et al., 2006, describe a 5-step differentiation protocol:
Stage 1 (substantially final endoderm production)
Stage 2 (substantially PDXl-negative foregut endoderm production)
Stage 3 (substantially PDXl-positive foregut endoderm production)
Stage 4 (substantially pancreatic endoderm, or epithelial or pancreatic endocrine precursor production), and stage 5 (substantially hormone-expressing endocrine cell production).
Importantly, for the first time, all these cell forms can be produced by the suspension methods described herein.

As used herein, "final endoderm (DE)" refers to pluripotent endoderm linenage cells that can differentiate into intestinal or intestinal-derived organ cells. According to one embodiment, the final endoleaf cells are mammalian cells. And in a preferred embodiment, the final endochlorous cell is a human cell. In some embodiments of the invention, the final endochlorous cells either express or do not significantly express certain markers. In some embodiments, one or more markers selected from SOXl7, CXCR4, MXLl, GATA4, HNF3beta, GSC, FGFl7, VWF, CALCR, FOXQl, CMKORl, CRIPL, and CER will be the final inner leaf. It is expressed in cells. In other embodiments, OCT4, α-fetoprotein (AFP), Thrombomodulin (Thrombomodulin).
One or more markers selected from registered trademarks)), SPARC, SOX7, and HNF4alpha are not significantly expressed in the final endoleaf cells. The final endoderm cell assembly and method of its production are described in U.S. Patent Application No. 11 / 021,618, filed December 23, 2004, named DEFINITIVE ENDODERM. The entire application is incorporated herein by reference.

Still different embodiments of the present invention relate to "PDXl-negative foregut foliage cells" and "foregut foliage cells", and cell cultures and cell aggregates of their equivalents. PDXl-negative foregut foliage cells are also pluripotent, with a variety of cells and the thymus, thyroid gland, parathyroid gland, lung / bronchus, liver, pharynx, gill sac, part of the duodenum and Eusterquio duct Can result in an organization containing. In some embodiments, the foregut endoderm cells are SOX17, HNFlB, HNFlα, FOXAl with non-foregut endoderm cells, eg, the final endoderm or PDX-positive endoderm that does not significantly express these markers. In comparison, it is expressed at increased levels. PDXl-negative foregut foliage cells express or do not express PDXl, AFP, SOX7, and SOXl at low levels. PDXl-negative foregut endoderm cell aggregates and methods of their production were filed on October 27, 2006 for PDXI-expressing dorsal and ventral foregut endoderm cells (PDXl-expressing dorsal and). It is described in US Patent Application No. 11 / 588,693 entitled ventral foregut endoderm), which is incorporated herein by reference in its entirety.

Other embodiments of the present invention relate to cell culture of "PDXl-positive pancreatic foregut endoderm cells" and "PDXl-positive prepancreatic endoderm" or equivalents thereof. PDXl-positive prepancreatic endofoliate cells are pluripotent and can result in a variety of cells and / or tissues including the stomach, intestine and pancreas. In some embodiments, PDXl-positive prepancreatic endochlorous cells increased PDXl, HNF6, SOX9, and PROXl as compared to non-prepancreatic endochlorous cells that did not express these markers in high amounts. Expressed at the level. Also, PDXl-positive prepancreatic endoleaf cells express NKX6.1, PTFlA, CPA, and cMYC at low levels or no expression at all.

Other embodiments of the present invention include "PDXl-positive pancreatic endoderm cells", "PDXl-positive pancreatic progenitor", and "pancreatic epithelium". ) ”,“ PE ”or their equivalents. PDXl-positive pancreatic progenitor cells are pluripotent and can result in a variety of cells in the pancreas, including acinars, ducts and endocrine cells. In some embodiments, PDXl-positive pancreatic progenitor cells express PDXl and NKX6.1 at increased levels compared to non-prepancreatic endoleaf cells that do not express these markers in high amounts. Also, PDXl-positive pancreatic progenitor cells express or do not express PTFlA, CPA, cMYC, NGN3, PAX4, ARX, NKX2.2, INS, GCG, GHRL, SST, and PP at low levels.

Alternatively, another embodiment of the present invention relates to cell culture of "PDXl-positive pancreatic endoderm tip cells" or an equivalent thereof.
In some embodiments, PDXl-positive pancreatic endoderm end cells express increased levels of PDXl and NKX6.1, similar to PDXl-positive pancreatic progenitor cells, unlike PDXl-positive pancreatic progenitor cells. PDXl-positive pancreatic endoderm progenitor cells further express PTFlA, CPA and cMYC at increased levels. PDXl-positive pancreatic endoderm end cells express or do not express NGN3, PAX4, ARX and NKX2.2, INS, GCG, GHRL, SST, and PP at low levels or at all.

Other embodiments of the present invention relate to cell culture of "pancreatic endocrine progenitor cells", "pancreatic endocrine progenitor cells" or their equivalents. Pancreatic endocrine progenitor cells are pluripotent and result in mature endocrine cells, including alpha, beta, delta and PP cells. In some embodiments, pancreatic endocrine progenitor cells express NGN3, PAX4, ARX, and NKX2.2 at increased levels as compared to other non-endocrine progenitor cell types. Also, pancreatic progenitor cells express low levels or no expression of INS, GCG, GHRL, SST, and PP.

Further embodiments of the present invention are obtained in vitro from pluripotent cells with respect to cell culture of "pancreatic endocrine cells", "pancreatic hormone secreting cells", "pancreatic islet hormone expressing cells", or equivalents thereof. With respect to cells such as alpha, beta, delta and / or PP cells or combinations thereof. Endocrine cells can be poly-hormonal or single-hormonal, eg, insulin, glucagon, ghrelin, somatostatin, and pancreatic polypeptides or combinations thereof. Therefore, endocrine cells can express one or more pancreatic hormones. It has at least some of the functions of human pancreatic cells. Islet hormone-expressing cells can be mature or immature. Mature pancreatic islet hormone-expressing cells and immature pancreatic islet hormones based on differential expression of certain markers or based on in vitro or in vivo functional capacity, such as glucose reactivity. Expressive cells can be distinguished. Also, pancreatic endocrine cells express or do not express NGN3, PAX4, ARX, and NKX2.2 at low levels.

Most of the above cell forms are epithelialized as compared to the final mesenchymal inner leaf cells. In some embodiments, the pancreatic endofollicles are PDXl EXPRESSING DOSAL AND VENTRAL, filed on October 27, 2006, for PDXI-expressing dorsal and ventral anterior intestinal flora.
One or more markers selected from Table 3 and / or one or more markers selected from Table 4 in U.S. Patent Application 11 / 588,693 under the name FOREGUT ENDODERM), and April 2005. It expresses one or more markers of US patent application 11/115,868, named PDXl-expressing endoderm, filed on the 26th. These are incorporated herein by reference in their entirety.

The present invention contemplates compositions and methods useful for differentiating cells, regardless of their source or their plasticity. As used herein, the "formability" of cells is used in a general sense, as is the case in the art. That is, cell formation refers to the ability of cells to differentiate into specific cell types found in tissues or organs from a fetal, fetal or grown organism. The more "formable" a cell is, the more tissue the cell may be able to differentiate. A "pluripotent cell" includes cells and their progeny. They differentiate into or result in pluripotent, multipotent, oligopotent, and unipotent cells, and / or a few if not all. It becomes a mature or partially mature cell type found in an organ from a foetation, fetal or grown organism. A "multi-potency cell" contains cells and their progeny. They differentiate into multi-differentiating, oligopotent, and unipotent cells, result in them, and / or mature or partially mature cell types are limited to specific tissues, organs, or organ systems. Except as the case, it becomes one or more mature or partially mature cell types. For example, multi-potency hematopoietic progenitor cells and / or their progenitors have the ability to differentiate into or yield to oligopotent cells such as one or more types of myeloid progenitor cells and lymphoid progenitor cells. It also results in other mature cell compositions usually found in the blood. An "oligopotent cell" includes cells having a more limited ability to differentiate into mature or partially mature cells than pluripotent cells and their progeny. However, oligopotent cells are still capable of differentiating into oligopotent and unipotent cells and / or specific tissues, organs or organ systems of one or more mature or partially mature cell types. be able to. One example of oligopotent cells is myelogenous progenitor cells. It can result in mature or partially mature erythrocytes, platelets, basophils, eosinophils, neutrophils and mononuclear cells. A "unipotent cell" is a cell and those that have the ability to differentiate into or bring about other monopoly cells and / or one type of mature or partially mature cell type. Includes descendants of.

The differentiateable cells used herein can be pluripotent, multipotent, oligopotent, or unipotent. In one embodiment of the invention, the differentiateable cell is a pluripotent, differentiable cell. In a more specific embodiment, pluripotent and differentiable cells are selected from the group consisting of yes stem cells, ICM / blastoderm superior cells, primordial ectoderm cells, primordial germ cells, and malformed cancer cells. In one particular embodiment, the differentiateable cell is a mammalian yes stem cell. In a more specific embodiment, the differentiateable cell is a human embryonic stem cell.

The present invention also contemplates differentiateable cells from any source in an animal, provided that the cells are differentiateable as defined herein. For example, differentiateable cells can be obtained from more mature tissues such as the fetal or any primordial germline, placenta or perineal tissue within it, or adult stem cells, such as lipids, bone marrow. Can be obtained from, but is not limited to, nerve tissue, breast tissue, liver tissue, pancreatic tissue, epithelial tissue, respiratory tissue, gonad and muscle tissue. In a specific embodiment, the differentiateable cell is a yes stem cell. In another specific embodiment, the differentiateable cell is an adult stem cell. Furthermore, in another specific embodiment, the stem cell is a stem cell derived from the placenta or chorion.

The present invention, of course, contemplates the use of differentiateable cells from any animal capable of developing differentiable cells. The animal from which the differentiateable cells are obtained can be a vertebrate, an invertebrate, a mammal, a non-mammal, a human, or a non-human. Examples of animal sources include, but are not limited to, primates, rodents, canines, felines, horses, bovids, and bovids.

Differentiable cells of the invention can be induced by using any method known to those of skill in the art. For example, human pluripotent cells can be produced by using dedifferentiation and nuclear transplantation methods. Furthermore, the human ICM / blastoderm upper layer cells or primordial ectoderm cells used in the present invention can be induced in vivo or in vitro. Primitive ectoderm cells can be produced as cell aggregates in adherent or suspension cultures, as described in WO 99/53021. In addition, human pluripotent cells can be passaged by any method known to those of skill in the art, including manual passage methods and bulk passage methods such as enzymatic or non-enzymatic passage methods. Can be used.

If embryonic stem cells are utilized in some embodiments, the yes stem cells have a normal karyotype, whereas in other embodiments, the yes stem cells have an abnormal karyotype. In one embodiment, the majority of yes stem cells have a normal karyotype. 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more are normal in the middle stage of mitosis examined. It is intended to show karyotype.

In another embodiment, the majority of yes stem cells have an abnormal karyotype. 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more are abnormal in the middle stage of mitosis investigated. It is intended to show karyotype. In certain embodiments, abnormal karyotypes are apparent after 5 or more, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 cells have been subcultured. In one specific embodiment, the abnormal karyotype comprises at least one autosomal trisomy in which the autosomal chromosome is selected from the group consisting of chromosomes 1, 7, 8, 12, 14 and 17. In another embodiment, the abnormal karyotype comprises three autosomal chromosomes, one of which is more than one and more than one selected from the group consisting of chromosomes 1, 7, 8, 12, 14 and 17. .. In one embodiment, the autosomal chromosome is chromosome 12 or 17. In another embodiment, the abnormal karyotype further comprises a sex chromosome. In one embodiment, the karyotype comprises two X chromosomes and one Y chromosome. It is also assumed that chromosomal translocations may occur, and such translocations are included in the term "abnormal karyotype". In addition, the combination of the above chromosomal abnormality and other chromosomal abnormality is included in the present invention.

The compositions and methods of the present invention include basal nutrient solutions. As used herein, a basal nutrient solution is a mixture of certain bulk inorganic ions that are essential for normal cell metabolism and salts that provide the cells with water, intracellularly and. A mixture of salts that maintains an extracellular osmotic balance, provides carbohydrates as an energy source, and provides a buffering system for maintaining the vehicle within the physiological pH range. Examples of basal nutrient solutions are Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMl1640, Ham's F-10 (Ham's). F-10),
Ham's F-12, Alpha-Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscob's Improved Dulbecco Medium (G-MEM). Iscove's Modified Dulbecco's Medium), and mixtures thereof, but is not limited to these. In one specific example, the basal nutrient solution is a mixture of DMEM and Hams F12 at about 50:50.

It is contemplated that the composition will further contain trace elements. Trace elements can be purchased commercially, for example from Mediatech. Non-limiting examples of trace elements include, but are not limited to, aluminum, chlorine, sulphate, iron, cadmium, cobalt, chromium, germanium, sodium, potassium, calcium, phosphate and magnesium. Specific examples of compounds containing trace elements include AlCl ₃ , silver nitrate, Ba (C ₂ H ₃ O ₂ ) ₂ , CdCl ₂ , CdSO ₄ , CoCl ₂ , CrCl ₃ , Cr ₂ (SO ₄ ) ₃ , CuSO. ₄ , ferric citrate, GeO ₂ , KI, KBr, LI, molybdic acid, MnSO ₄ , MnCl ₂ , NaF, Na ₂ SiO ₃ , NaVO ₃ , NH ₄ VO ₃ , (NH ₄ ) ₆ Mo ₇ O ₂₄ , NiSO ₄ , RbCl, selenium, Na ₂ SeO ₃ , H ₂ SeO ₃ , selenate-2Na, selenomethionone, SnCl ₂ , ZnSO ₄ , ZrOCl ₂ , mixtures thereof, and salts thereof. It is not limited to. In the presence of selenium, selenate or selenomethionone, it is present at a concentration of about 0.002 to about 0.02 mg / L. In addition, hydroxylapatite can also be present.

It is contemplated that amino acids can be added to the defined medium. Non-limiting examples of such amino acids include glycine, L-alanine, L-alanyl-L-glutamine, L-glutamine / glutamine, L-arginine hydrochloride, L-asparagine-H ₂ O, L-. aspartic acid, L- cysteine hydrochloride H ₂ O, L- cystine 2HCl, L- glutamic acid, L- histidine hydrochloride _-H 2 O, L- isoleucine, L- leucine, L- lysine hydrochloride, L- methionine, L- Examples include phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium dihydrate, and L-valine. In certain embodiments, the amino acid is L-isoleucine, L-phenylalanine, L-proline, L-hydroxyproline, L-valine, and mixtures thereof.

It is also contemplated that the defined medium can contain ascorbic acid. Desirably, ascorbic acid is from about 1 mg / L to about 1000 mg / L, or 2 mg / L to about 500 mg / L, or about 5 mg / L to about 100 mg / L, or about 10 mg / L to about 100 mg / L, or about. It is present at an initial concentration of 50 mg / L.

In addition, the compositions and methods of the invention can include other components such as serum albumin, iron-binding globulin, L-glutamine, lipids, antibiotics, β-mercaptoethanol, vitamins, minerals, ATP, and the like. Can exist. Examples of vitamins that can be present are vitamins A, B ₁ , B ₂ , B ₃ , B ₅ , B ₆ , B ₇ , B ₉ , B ₁₂ , C, D ₁ , D ₂ , D ₃ , D. _{4, D} 5, E, tocotrienols, including _{K 1} and _{K 2,} are not limited to. One of ordinary skill in the art can determine the optimum concentration for use in a particular culture, such as minerals, vitamins, ATP, lipids, essential fatty acids, etc. For example, the concentration of the supplement can be from about 0.001 microM to about 1 mM or more. Specific examples of concentrations at which the supplement can be provided are approximately 0.005 microM, 0.01 microm, 0.05 microm, 0.1 microm, 0.5 microm, 1.0. Examples thereof include micro M, 2.0 micro M, 2.5 micro M, 3.0 micro M, 4.0 micro M, 5.0 micro M, 10 micro M, 20 micro M, and 100 micro M. Not limited to these. In one specific embodiment, the compositions and methods of the invention comprise vitamin B6 and glutamine. In another specific embodiment, the compositions and methods of the invention include vitamin C and iron supplements. In another specific embodiment, the compositions and methods of the invention comprise Vitamin K ₁ and Vitamin A. In another specific embodiment, the compositions and methods of the present invention include vitamin D ₃ and ATP. In another specific embodiment, the compositions and methods of the present invention includes a vitamin B ₁₂ and iron-binding globulin. In another specific embodiment, the compositions and methods of the invention include tocotrienols and β-mercaptoethanol. In another specific embodiment, the compositions and methods of the invention comprise glutamine and ATP. In another specific embodiment, the compositions and methods of the invention comprise omega-3-fatty acids and glutamine. In another specific embodiment, the compositions and methods comprise an omega 6 fatty acids and vitamin B _1. In another specific embodiment, the compositions and methods of the invention include α-linolenic acid, and B ₂ .

The compositions of the present invention are virtually serum-free. As used herein, "substantially serum-free" refers to the absence of serum in the solutions of the invention. Serum is not an essential ingredient in the compositions and methods of the invention. Thus, the presence of serum in all compositions is simply an impurity, eg, residual formation from cell primary cultures or from starting material. For example, a medium or environment that is virtually serum-free should contain less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% serum. And the improved ability of the medium or environment to maintain bioactivity is still observed. In a specific embodiment of the invention, a composition that is virtually serum-free does not contain serum or serum substitute, or from isolation of serum or serum substitute component added to a defined medium. Contains only trace amounts of serum or serum substitutes.

The compositions and methods of the invention include means of stimulating ErbB2 tyrosine kinase activity in differentiateable cells. In a specific embodiment of the invention, the compositions and methods of the invention include the presence of at least one ErbB3 ligand. Typically, the ErbB3 ligand binds to the ErbB3 receptor and dimerizes with the ErbB2 receptor. The ErbB2 receptor then becomes responsive to intracellular tyrosine kinase activity in differentiateable cells.

As used herein, an "ErbB3 ligand" is a ligand that binds to ErbB3 and then dimerizes with ErbB2, resulting in the tyrosine kinase activity of the ErbB2 portion of the ErbB2 / ErbB3 heterodimer receptor. It means something that activates. Non-limiting examples of ErbB3 ligands include neuregulin-1; neuregulin-1 splice variants and isoforms, including HRG-β, HRG-α, and Neu Differentiation Factor (NDF), and acetylcholine receptors. Includes Origin Action (ARIA), Glycer Growth Factor 2 (GGF2), and Sensory And Motor Neuron-Derived Factor (SMDF), Neuregulin-2; NRG2-β. Examples include, but are not limited to, splice variants and isoforms of neuregulin-2; epiligulin; biregulin.

In one embodiment, the means for stimulating ErbB2-derived tyrosine kinase activity are neuregulin-1, heregulin-β (HRG-β), hellegulin-α (HRG-α), new differentiation factor (NDF). ), Receptor-derived activator of acetylcholine (ARIA), collagen cell growth factor 2 (GGF2), motor-neuron-derived factor (SMDF), neuregulin-2, neuregulin-2β (NRG2-β) , Epiregulin, neuregulin, and variants thereof, comprising at least one ErbB3 ligand selected from the group consisting of functional fragments. In another specific embodiment, the compositions and methods of the invention include, for example, more than one means of stimulating ErbB2-derived tyrosine kinase activity, including but not limited to the use of two or more ErbB3 ligands. Including.

In a more specific embodiment, the compositions and methods of the invention are that the ErbB3 ligand is HRG-β, or a variant thereof, a functional fragment. In one embodiment, the functional fragments of media-added proteins, polypeptides, variants or derivatives thereof are the same as the cell type being cultured. For example, when mouse embryonic stem cells are cultured, H has the same amino acid sequence as the mouse (mus musculs) HRG-β sequence as an additive in the culture.
RG-β can be used and is considered to be "same species". In other embodiments, the species derived from the biological additive is considered to be different from the cells to be cultured. For example, when mouse embryonic stem cells are cultured, HRG-β having the same amino acid sequence as the human HRG-β sequence can be used and is considered to be a "different species".

As used herein, "functional fragment" refers to a fragment or splice variant of a full-length polypeptide that produces similar physiological or cellular effects as a full-length polypeptide. .. The biological effect of a functional fragment need not be in the same range or intensity as a full-length polypeptide, as long as similar physiological or cellular effects are seen. For example, a functional fragment of HRG-β can detectably stimulate ErbB2-derived tyrosine kinase.

As used herein, the term "mutant" includes chimeric or fused polypeptides, homologues, analogs, and orthologs, or paralogs. Further, a variant of the referenced protein or polypeptide is a protein or polypeptide whose amino acid sequence is at least about 80% identical to the referenced protein or polypeptide. In a specific embodiment, the variant is at least about 85%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% identical to the comparative protein or polypeptide. When the term "corresponding" is used in the context of sequence alignment herein, it is intended to mean a described position within a referenced protein or polypeptide. For example, wild-type human or mouse neuregulin-1 and its position in the referenced protein or polypeptide in a modified protein or polypeptide. Thus, when the amino acid sequence of the protein or polypeptide of interest is aligned with the amino acid sequence of the referenced protein or polypeptide sequence, the sequence corresponding to the stated position of the amino acid sequence of the referenced protein or polypeptide. Is an array that is aligned with the position of the referenced array and does not have to be in the exact same position as the reference array. The method of arranging the sequences for determining the corresponding amino acids between the sequences will be described below.

A polypeptide having, for example, at least about 95% "same" amino acid sequence as the referenced amino acid sequence code, eg TGF-β, is 100 of the reference amino acid sequence encoding TGF-β referenced by the amino acid sequence of the polypeptide. It is understood to mean that the amino acid sequence of a polypeptide is identical to the reference sequence, except that it can contain up to approximately 5 modifications per amino acid. In other words, to obtain a peptide that has about 95% the same amino acid sequence as the reference amino acid sequence, up to about 5% of the amino acid residues in the reference sequence can be deleted or replaced with other amino acids, or referenced. Up to about 5% of total amino acids can be inserted into the sequence. These modifications of the reference sequence can occur at the N- or C-terminal positions of the reference amino acid sequence, or anywhere between their terminal positions, and can be present individually between the amino acids of the reference sequence or the reference sequence. It can exist as one or more adjacent groups within.

As used herein, the term "identity" is a measure of nucleotide or amino acid sequence identity compared to the referenced nucleotide or amino acid sequence. In general, the sequences are arranged so that the highest order of match is obtained. "Identity" itself has a meaning recognized in the art and can be calculated by known art. (See: eg, Computerized Molecular Biology, Lesk, AM.
, Ed. , Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D.M. W. , Ed. , Academymic
Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. et al. M. ， And
Griffin, H. et al. G, eds. , Humana Press, New Jersey (1994); von Heinje, G, Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. et al. and De
vereux, J. et al. , Eds. , M Stockton Press, New York (1991)). Although there are several methods for measuring identity between two polynucleotide or polypeptide sequences, the term "identity" is known to those of skill in the art (Carillo, H. & Lipton, D. et al. , Siam J Applied Math 48: 1073 (1988)). The methods commonly used to determine the identity or similarity between two sequences are, but are not limited to, Guide to Huge Computers, Martin J. et al. Bishop, ed. , Academ
ic Press, San Diego (1994) and Carillo, H. et al. & Lipton, D.I. , Siam J Applied Math 48: 1073 (1988). Computer programs can include methods and algorithms for calculating identity and similarity. Examples of computer programming methods for determining identity and similarity between two sequences are, but are not limited to, GCG program packages (Deverex, J., et al., Nucleic Acids Research 12 (i):. 387 (1984)), BLASTP, ExPASy5 BLASTN, FASTA (Atschul, SF, et al., J Program Biol 215: 403 (1990)) and FASTA DB. Examples of methods for determining identity and similarity are described in Michaels, G. et al. and Galian, R.M. , Current Proteins in Protein Science, VoI 1, John Wiley & Sons, Inc. (2000), which is referenced and incorporated herein. In one embodiment of the invention, the algorithm used to determine identity between two or more polypeptides is BLASTP.

In another embodiment of the invention, the algorithm used to determine identity between two or more polypeptides is FASTDB. This is based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1990)). This is referenced and incorporated herein. In the FASTDB sequence alignment, the control sequence is an amino sequence. The identity of the resulting sequence alignment is expressed as a percentage. The parameters used for the FASTDB alignment of amino acid sequences to calculate the percentage of identity are, but are not limited to: Matrix = PAM, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Win Doe size = 500 or the length of the amino sequence of interest, whichever is shorter.

If the sequence of interest is shorter or longer than the sequence of interest due to N-terminal or C-terminal additions or chromosomal deletions, manually, whether due to internal additions or chromosomal deletions. Can be corrected. This is because the FASTDB program does not calculate the N-terminal and C-terminal truncations or additions of the sequence of interest when calculating for percentage identity. For sequences that are truncated at the 5'or 3'end with respect to the sequence of interest, the percentage of identity is the base of the target sequence that is N-terminal and C-terminal to the unmatched / unaligned target sequence. By calculating for the number of, it is modified as a percentage of the total bases of the target sequence. The result of the FASTDB sequence alignment determines the matching / alignment. The percentage of alignment is subtracted from the percent identity calculated in the FASTDB program above using specific parameters to obtain the final percent identity score. This modified score can be used as well as the percent identity for the purpose of determining how the alignments "correspond" to each other. For the purpose of manually adjusting the percent identity score, the residues of the question (subject) sequence that extend beyond the N- or C-terminus of the reference or subject sequence can be considered. That is, residues that are not matched / aligned to the N- or C-terminus of the comparison sequence can be calculated when manually adjusting the percent identity score or alignment numbering.

For example, a 90 amino acid residue test sequence is aligned with a 100 residue reference sequence to determine percent identity. Chromosome deletion occurs at the N-terminus of the sequence of interest. Therefore, the FASTDB sequence does not show a match / alignment of the first 10 residues at the N-terminus. From the percent identity score calculated by the FASTDB program when 10 unpaired residues represent 10% of the sequence (when the number of residues at the N- and C-terminus does not match the total number of residues in the test sequence). 10% is subtracted. If the remaining 90 residues are perfectly matched, the final percent identity is 90%. In another example, the 90 residue subject sequence is compared to the 100 subject sequence. At this time, the chromosomal deletion is an internal deletion that has no residue at the N- or C-terminus of the target sequence, does not match the target, or is not aligned. In this case, the percentage identity calculated by FASTDB is not manually corrected.

The present invention also provides chimeric or fusion polypeptides. As used herein, a "chimeric polypeptide" or "fusion polypeptide" includes at least some of the members of a reference polypeptide that are effectively linked to a second different polypeptide. The second polypeptide has an amino acid sequence corresponding to a polypeptide that is not substantially the same as the reference polypeptide, and the polypeptide is derived from the same or different organisms. With respect to the fusion polypeptide, the term "effectively link" is adapted so that the reference polypeptide and the second polypeptide fuse with each other to achieve the expected function that occurs in the sequences in which both sequences are used. Intended to be. A second polypeptide can be fused to the N-terminus or C-terminus of the reference polypeptide. For example, in one embodiment, the fusion polypeptide is a GST-IGF-1 fusion polypeptide in which the IGF-1 sequence is fused to the C-terminus of the GST sequence. Such fusion polypeptides can facilitate the purification of recombinant polypeptides. In another embodiment, the fusion polypeptide can comprise a heterologous signal sequence at the N-terminus. In certain host cells (eg, mammalian host cells), expression and / or secretion of the polypeptide can be increased by the use of a heterologous signal sequence.

In addition to fragment and fusion polypeptides, the present invention includes homologues and analogs of naturally occurring polypeptides. A "homlogue" is defined herein as two nucleic acids or polypeptides having similar or identical nucleotide or amino acid sequences. Homologues include allelic polymorphisms, orthologs, paralogs, agonists, and antibodies, as defined below. The term "homlogue" further includes nucleic acid molecules that differ from the reference nucleotide sequence due to the degeneracy of the genetic code and thus encode the same polypeptide as encoded by the reference nucleotide sequence. As used herein, "naturally occurring" refers to a naturally occurring nucleic acid or amino acid sequence.

A polypeptide agonist can retain substantially the same or a subset of the polypeptide's biological activity. An antagonist of a polypeptide can ban one or more of the activities naturally occurring from the polypeptide.

In a more specific embodiment of the compositions and methods of the invention, the ErbB3 ligand is HRG-β, a variant or functional fragment thereof. In addition, non-limiting examples of ErbB3 ligands are disclosed below. U.S. Patent No. 6,136,558, 6,387,638, and 7,063,961. These are incorporated herein by reference.

In general, heregulin is classified into two major types, alpha and beta, based on the EGF-like domains of two variants that differ at the C-terminal portion. However, these EGF-like domains have the same spacing of the six cysteine residues contained therein. Based on amino acid sequence comparisons, Holmes et al. Found that among the 1st to 6th cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB EGF), and ampphiregulin ( We found that it was 35% identical to AR), 32% identical to TGF-α, and 27% identical to EGF.

44KDa New Differentiation Factor (NDF) is a rat equivalent of human HRG. Like the HRG polypeptide, NDF lacks the N-terminal signal peptide, with the EGF-like domain following the immunoglobulin (Ig) homology domain. Currently, there are at least 6 different fibroblast pro-NDFs classified as either alpha or beta polypeptides based on the sequence of the EGF-like domain. Isoforms 1-4 are characterized on the basis of varying stretches between the EGF-like domain and the transmembrane region. Therefore, different NDF isoforms appear to be generated by changes in splicing and can perform different tissue-specific functions. See EP 505148; WO 93/22424; and WO 94/28133. These are incorporated herein by reference.

In one embodiment of the invention, the compositions and methods of the invention do not include exogenous insulin and insulin substitutes. The phrase "not including extrinsic insulin or insulin substitute" is used herein to indicate that insulin or insulin substitute is not deliberately added to the compositions or methods of the invention. In certain embodiments of the invention, the methods and compositions of the invention therefore do not include insulin or insulin substitutes that are deliberately supplied. However, this does not mean that the compositions or methods of the invention cannot necessarily step on endogenous insulin. As used herein, "endogenous insulin" indicates that cultured cells can produce insulin as their own action when cultured by the methods of the invention. Endogenous insulin may also be used to indicate impurities from primary cell cultures or residual impurities from starting materials. In a specific example, the compositions and methods of the invention are 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. Contains less than micro g / mL insulin.

As used herein, the term "insulin" refers to a protein, variant or fragment thereof that can bind to an insulin receptor at normal physiological concentrations and elicit a signal through the insulin receptor. The term "insulin" includes all natural human insulins, other mammalian insulins, proteins having polypeptide sequences of homologues or variants of these sequences. In addition, the term insulin includes a polypeptide fragment that can bind to the insulin receptor and trigger a signal through the insulin receptor. The term "insulin substitute" also includes all zinc-containing compounds used in place of insulin to give substantially similar results as insulin. Examples of insulin substitutes include, but are not limited to, zinc chloride, zinc nitrate, zinc bromide, and zinc sulphate.

To be clear, insulin-like growth factor is neither an insulin substitute intended in the present invention nor a homologue of insulin. Thus, in another specific embodiment, the compositions and methods of the invention include the use of at least one insulin-like growth factor (IGF), variant or functional fragment thereof. In another embodiment, the compositions and methods of the invention also do not include all extrinsic insulin-like growth factors (IGFs). In a specific embodiment, the compositions and methods of the invention are 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 ng / Contains less than mL IGF-1.

As used herein, the term "IGF-1R activator" refers to a mitogen that plays a vital role in regulated cell proliferation, differentiation, and apoptosis. The effect of the activator of IGF-1R can be mediated through other receptors, but is usually mediated through IGF-1R. IGF-1R also contains cell transformations caused by oncovirus proteins and oncogene products, and interactions are regulated by a group of specific binding proteins (IGFBPs). In addition, a large group of IGFBP proteases hydrolyze IGFBPs, dissociate IGFs bonds, and resume their ability to interact with IGF-1R. For the purposes of the present invention, ligands, receptors, binding proteins, and proteases are all considered to be activators of IGF-1R. In certain embodiments, the activator of IGF-1R is IGF-1, or IGF-2. In a further embodiment, the activator of IGF-1R is an IGF-1 analogue. Non-limiting examples of IGF-1 analogs are LongR3-IGFl, Des (l-3) IGF-1, [Arg ³ ] IGF-l, [Ala ³¹ ] IFG-1, Des (2, 3) [ Ala ³¹ ] IGF-1, [Leu ²⁴ ] IGF-l, Des (2,3) [Leu ²⁴ ] IGF-l, [Leu ⁶⁰ ] IGF-l, [Ala ³¹ ] [Leu ⁶⁰ ] IGF-l, [ Leu ²⁴ ] [Ala ³¹ ] IGF-l, and combinations thereof. In a further embodiment, the IFG-1 analog is LongR3-IGFl. LongR3-IGFl is a recombinant analog of human insulin growth factor-1. LongR3-IGFl is from about 1 ng / mL to about 1000 ng / ml, more preferably from about 5 ng / ml to about 500 ng / mL, more preferably from about 50 ng / ml to about 500 ng / mL, more preferably from about 100 ng / ml to about 100 ng / ml. It is assumed to be initially present at a concentration of 300 ng / mL, or about 100 ng / ml.

In certain embodiments, the compositions and methods of the invention comprise transforming growth factor beta (TGF-β), or the TGF-β family, variants or functional fragments thereof. As used herein, the term "member of the TGF-β family" or similar terms used in connection with growth factors are commonly used by those skilled in the art associated with the TGF-β family. It is characterized by the homology known to one or a member of the TGF-β family, or the similarity of function known to a member of the TGF-β family. In a specific embodiment of the invention, in the presence of members of the TGF-β family, members of the TGF-β family, variants or functional fragments thereof activate SMAD2 or 3. In certain embodiments, members of the TGF-β family consist of a group consisting of Nodal, activin A, activin B, TGF-β, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-4 (BMP4). To be elected. In one embodiment, the member of the TGF-β family is activin A.

In the presence of Nodal, from about 0.1 ng / mL to about 2000 ng / mL
It may be present in ml, more preferably from about 1 ng / ml to about 1000 ng / mL, more preferably from about 10 ng / ml to about 750 ng / mL, and more preferably from about 25 ng / ml to about 500 ng / mL. It is planned. When used, Actibin A is from about 0.01 ng / mL to about 1000 ng / ml, more preferably from about 0.1 ng / ml to about 100 ng / mL, more preferably from about 0.1 ng / ml to about 25 ng / ml. It is intended to be present at an initial concentration of mL, or preferably about 10 ng / ml. If present, TGF-β is from about 0.01 ng / ml to about 100 ng / mL, more preferably from about 0.1 ng / ml to about 50 ng / mL, more preferably from about 0.1 ng / ml to about 20 ng / mL. It is intended to be present at an initial concentration of mL.

In an additional embodiment of the invention, the compositions and methods of the invention do not include activators of FGF receptors. Currently, there are at least 22 known members of the family of fibroblast growth factors, which bind to at least one of the four FGF receptors. As used herein, the term "FGF receptor activator" is commonly known to those of ordinary skill in the art associated with the FGF-β family, or to members of the FGF-β family. A growth factor characterized by homology or functional similarity known to members of the FGF-β family. In certain embodiments, the activator of the FGF receptor is FGF, such as, but is not limited to, αFGF and FGF2. In a specific embodiment, the composition and method do not include exogenous FGF2. The term "external FGF2 free" is used herein as an indication that fibroblast growth factor 2, i.e. basic FGF, is not deliberately added to the compositions or methods of the invention. Therefore, in certain embodiments of the invention, the methods and compositions of the invention do not contain deliberately supplied FGF2. However, it does not necessarily mean that the compositions or methods of the invention cannot contain endogenous FGF2. As used herein, "endogenous FGF2" indicates that the cultured cells themselves can produce FGF2 when cultured by the methods of the invention. Endogenous FGF2 may also be used to indicate impurities from primary cell cultures or residual impurities from starting materials. As a specific example, the composition or method of the present invention comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or FGF2 of 1 ng / mL or less.

However, it is contemplated that the compositions and methods of the invention can include at least one activator of a FGF receptor, including a FGF polypeptide, a functional fragment thereof or a variant thereof. In the presence of FGF2, from about 0.1 ng / mL to about 100 ng / ml, more preferably from about 0.5 ng / ml to about 50 ng / mL, more preferably from about 1 ng / ml to about 25 ng / mL, more preferably. Is about 1 ng / ml to about 12 ng / m
It is intended to be present at an initial concentration of L, and most preferably about 8 ng / ml. In another specific embodiment, the compositions and methods of the invention can comprise at least one activator of a FGF receptor other than FGF2. For example, the compositions and methods of the invention can include at least one of FGF-7, FGF10, FGF-22, variants thereof or functional fragments thereof. In specific embodiments, there are at least two of FGF-7, FGF-10 and FGF-22, variants thereof or functional fragments thereof. In another embodiment, all three are present: FGF-7, FGF-10, FGF-22, variants thereof or functional fragments thereof. If any of FGF-7, FGF-10, FGF-22, variants thereof or functional fragments thereof are present, each is from about 0.1 ng / mL to about 100 ng / ml, more preferably. At an initial concentration of about 0.5 ng / ml to about 50 ng / mL, more preferably about 1 ng / ml to about 25 ng / mL, more preferably about 1 ng / ml to about 12 ng / mL, and most preferably about 8 ng / ml. It is intended to exist.

In a further embodiment, the compositions and methods of the invention comprise serum albumin (SA). In a specific embodiment, the SA is either bovine SA (BSA) or human SA (HAS). In a specific embodiment, the concentration of SA is about 0.2% or more by volume (v / v), but is about 10% v / v or less. In a more specific embodiment, the concentration of SA is about 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 0.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3 0.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5 0.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7 0.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9 Higher than 0.0%, 9.2%, 9.4%, 9.6% and 9.8% (v / v).

In additional embodiments, the compositions and methods of the invention comprise at least one insoluble substrate. For example, but not limited to these, differentiated cells can be placed on the cell culture surface containing compounds such as polystyrene, polypropylene, and then the surface can be coated with an insoluble substrate. In a specific embodiment, the insoluble substrate is selected from the group consisting of collagen, fibronectin, fragments thereof or variants thereof. Other examples of insoluble substrates include, but are not limited to, fibrin, elastin, fibronectin, laminin, and nidgen.

Thus, the cell culture environment and methods of the present invention include plating cells in adhesive culture. As used herein, the terms "plated" and "plating" refer to any process that allows cells to grow in adherent cultures. As used herein, the term "adhesive culture" is a culture system in which cells are cultured on the surface of an individual, the surface of which is coated with an insoluble substrate, followed by a substrate as exemplified below. It is coated with another surface coating, or with any other chemical or biological substance that allows the cells to grow or stabilize. The cells can or may not adhere firmly to a solid surface, or a substrate. Hypokeimenons for adhesive culture are polyornithine, laminin,
One or any combination of polylysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycan, polysaccharide-degrading acid (PGA), polylactic acid (PLA), and polylactic acid glycolic acid (PLGA). Can be included. In addition, the substrate for adhesive culture can include a feeder layer or a matrix containing pluripotent human cells or cell cultures. As used herein, the term "extracellular matrix" includes, but is not limited to, the solid substrates described above, as well as feeder layers or pluripotent human cells or cell cultures. Includes matrix. In one embodiment, cells are plated on a plate coated with MATRIGEL®. In another embodiment, cells are plated on a fibronectin-coated plate. In one embodiment, when cells are plated on fibronectin, the plate is prepared with a coating of 10 microg / mL human plasma fibronectin (In vitrogen, # 33016-015), diluted with tissue grade water and at room temperature. Is held for 2-3 hours. In another embodiment, the serum can be placed in the medium for up to 24 hours to allow the cells to be plated on the plastic. If serum is used to promote cell adhesion, then the medium is transferred and a virtually serum-free composition is added to the plated cells.

The compositions and methods of the present invention contemplate that differentiateable cells are cultured under conditions that are essentially free of feeder cells or feeder layers. As used herein, a "feeder cell" is a cell that grows in vitro. That is, a cell that is co-cultured with a target cell and stabilizes the target cell at the stage of differentiation. As used herein, the term "feeder cell" and "feeder cell layer" can be used interchangeably. As used herein, the term "essentially free of feeder cells" refers to tissue culture conditions that do not contain feeder cells or contain a "de minimus" number of feeder cells. Is shown. The term "deminimum" means the number of feeder cells carried over from the previous culture conditions in which the differentiated cells were cultured on the feeder cells to the current culture conditions. In one embodiment of the above method, a conditioned medium is obtained from feeder cells that stabilize the target cells in the current state of differentiation. In another embodiment, the defined medium is unconditioned medium, which is not available from feeder cells.

As used herein, the term "stable" when used in connection with the cell differentiation state of a cell or cell culture means that the cell continues to grow in the medium for multiple generations, preferably medium. It continues to grow in, meaning that most, if not all, cells in the medium are in the same state of differentiation. When more stable cells divide, division usually results in cells of the same cell type or in the same differentiated state. In general, stable cells or cell populations do not further differentiate or redifferentiate if the culture conditions are not changed, the cells continue to be passaged and do not overgrow. In one embodiment, stable cells can proliferate in a stable state indefinitely or for at least two generations or more. In a more specific embodiment, the cells are in their 3s or higher, 4th or higher, 5th or higher, 6th or higher, 7th or higher, 8th or higher, 9th or higher, 10th or higher, 15th or higher, 20th or higher, The cells are stable for more than 25s and more than 30s. In one embodiment, the cells are about 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months, or 11 Stable in consecutive months of passage. In another embodiment, the cells are stable after about one year or more of continuous passage. In one embodiment, stem cells are maintained in a normal passage in a medium defined in a pluripotent state until they are desired to be differentiated. As used herein, the term "proliferation" refers to an increase in the number of cells in a cell culture.

In certain embodiments, the compositions and methods of the invention comprise an inactivator for BMP signaling. As used herein, "inactivator of BMP signaling" is antagonized by the activity of one or more BMP proteins, or either of their upstream or downstream signal components, via any of the possible signaling pathways. A drug to be used. The one or more compounds used for the activation of BMP signaling can be those known in the art or any compound discovered in the future. Non-limiting examples of inactivators for BMP signaling include dominant-negative, truncated BMP receptors, soluble BMP receptors, BMP receptor-Fc chimeras, noggin, follistatin ( follistatin, neurogenic factors (chordin), gremlin, cerberus / DAN family proteins, ventropin, high dose activin, and amnionless. ..

In certain embodiments, the compositions and methods of the invention can include at least one hormone, citkin, adipokine, growth hormone, variants thereof or functional fragments thereof. In certain embodiments, the growth hormone present in the defined medium is assumed to be of the same species as the differentiateable cells cultured in the defined medium. So, for example, if human cells are cultured, growth hormone is human growth hormone. It is also envisioned that growth hormone comes from a different species than the cultured cells. Desirably, hormones, citkins, adipokines, and / or growth hormone are from about 0.001 ng / ml to about 1000 ng / mL, more preferably from about 0.001 ng / ml to about 250 ng / mL, or more preferably about 0. It is present at an initial concentration of 0.01 ng / ml to about 150 ng / ml.

Examples of citkins and adipokines that can be included in the compositions and methods of the present invention include the 4α helix bundle family of citokines, the interleukin-1 (IL-I) family of citokins, the IL-17 family of citokines, And include, but is not limited to, the chemokine family of sitkins. Of course, the invention contemplates each of the members and subclass of these families of cytokines such as, for example, CC chemokines, CXC chemokines, C chemokines and CX 3 _C chemokines, interferons, interleukins, lymphotoxin acids , C-kit ligand, Granulocyte / Macrophage-Colony Stimulating Factor (GM-CSF),
Monocy-macrophage colony-stimulating factor M-CSF, granulocyte colony-stimulating factor (G-CSF), leptin, adiponectin, resistin, plasminogen activator inhibitor-1 (PAI-1), tumor necrosis Factor alpha (TNFα), tumor necrosis factor beta (TNFβ), leukemia inhibitory factor, bisfatin, retinol-binding protein 4 (RBP4), erythropoietin (EPO), thrombopoetin (THPO), but are limited to these. is not. Of course, one of ordinary skill in the art will appreciate that the present invention contemplates variants or functional fragments of the elements described above.

The present invention relates to a method of culturing differentiable cells, which comprises plating the differentiable cells on the surface of the cell culture, providing the cells with a basal nutrient solution to give the cells ErbB2-derived tyrosine. It involves providing a means of stimulating kinase activity.

In one embodiment, in the absence of serum or serum substitute, and in the absence of a feeder cell layer, at least one composition of the invention is in contact with the differentiateable cells and the cells are not present for at least 1 month. It is made to be maintained in a differentiated state. Pluripotency can be determined through cell characterization of surface markers, transcriptional markers, karyotypes, and the ability to differentiate into cells of the three germ layer layers. These characterizations are well known to those of skill in the art.

The cell aggregates described herein can be suspended in any physiologically acceptable medium and are typically selected according to the cell type contained. Tissue culture can include, for example, basic nutrients such as sugars and amino acids, growth factors, antibiotics (to minimize contamination), and the like. In another embodiment, the differentiateable cells are cultured in suspension using the cell culture medium described herein. When the term "suspension" is used in the context of cell culture, it is used in the sense used in the art. That is, the cell culture suspension is a cell culture environment in which cells or cell aggregates do not adhere to the surface. Those skilled in the art are familiar with suspension culture techniques, such as, but are not limited to, flow hoods, incubators, and / or devices used to keep cells in constant motion. For example, a stirrer platform, a shaker, and the like can be provided as needed. As used herein, a cell has "movement" if it is moving, or if the environment between them is moving relative to the cell. Once the cells are maintained in a "movement" state, in one embodiment the movements are designed to avoid or prevent the cells from being exposed to shear forces "gentle movements" or "gentle agitation". Hmm. "

Various methods of forming cell aggregates are known in the art, for example the "hanging drpo" method, in which cells are placed at the bottom of a drop in an inverted drop of tissue culture. Sink, gather, and shake the cell suspension in an experimental flask. Various variants of these techniques can also be used. For example, N. E. Timmins et al. (2004) Angiogenesis 7, 97-103; W. et al. Dai et al. (1996) Biotechnology and Bioengineering 50, 349-356; R. et al. A. Forty et al. (1996) Devopment 122, 1611-1620; Forgas et al., (2001) J. Mol. Biophyss. 74, 2227-2234 (1998); K.K. S. Fumkawa et al., Cell Transplantation 10, 441-445; Glicklis et al., (2004) Biotechnology and Bioengineering 86, 672-680; Carpenedo et al., (2007) Stem Cells 25, 2224-2234; and T. et al. Korff et al. (2001) FASEB J. et al. See 15,447-457. All of these are referenced and incorporated herein. More recently, cell aggregates scrape micropatterned colonies into suspensions and centrifuge microtiter plate colonies into suspensions, or use pipettes to pattern microwells. The colonies grown in (Ungrin et al., (2008) PLoS ONE 3 (2), 1-12; Bauwens et al., (2008), Stem Cells Published online June 26, 2008). Although such methods can be used to produce the cell aggregates described herein, the cell aggregates produced herein are for synchronous directed-differentiation. D above
Optimized as described in'Amour et al. 2006. Unlike these other methods, the methods for producing cell aggregates in suspensions described herein are susceptible to large scale production.

Generally, the cell medium composition of the present invention is refreshed at least once a day, but the medium can be changed more often or less depending on the specific needs and circumstances of the suspension culture. In vitro, cells are usually grown in culture in batch mode and exposed to a variety of media conditions. As described herein, cells are present in dish medium as adhesive cultures or as cell aggregates in suspension and come into contact with the surrounding medium; and the waste medium is replaced on a regular basis. Generally, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, The culture solution can be refreshed every 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or any time thereof. In additional examples, 1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days or 2 The medium can be refreshed every day or more, or for any period in between, but is not limited to these.

However, in another embodiment of the invention, a perfusion method is employed to prevent degradation of growth factors and other agents that must be replaced frequently, or from the medium over a period of time. The recirculation method was adopted as a means of depleting waste. For example, US Pat. No. 5,320,963 describes a bioreactor for perfusion culture of suspended cells. U.S. Pat. No. 5,605,822 describes a bioreactor system that uses stroma cells to provide growth factors for the growth of HSC cells in recirculation culture. U.S. Pat. No. 5,646,043 describes the growth of HSC cells in a continuous periodic recirculation containing a medium composition for the growth of HSC cells. U.S. Pat. No. 5,155,035 describes a bioreactor for suspension culture of cells by fluid medium rotation. These are referenced and incorporated herein by reference in their entirety.

Generally, cells suspended and cultured in the medium composition of the present invention are "split" or "passaged" almost weekly, but the cells have specific needs and suspension culture. It can be subcultured more frequently or less frequently, depending on the environment of the cell. For example, cells are 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or between them. Can be subrogated in any timeframe of. As used herein, when used in the context of cell culture, the terms "split" or "passage" are used in the sense used in the art. That is, cell culture split or passage is the collection of cells from a previous culture and the transfer of a smaller number of collected (harvested) cells to a new cell culture vessel. In general, passaged cells can continue to grow in a healthy cell culture environment. Those skilled in the art are familiar with the processes and methods of cell culture passage, and this is not always the use of enzymatic or non-enzymatic methods that can be used to separate cells that are aggregated together during their growth expansion. including.

In some cases, some cell death may occur in cells (suspended or adherently cultured) immediately after passage. In one embodiment, the differentiateable cells can be "recovered" from passage by delaying the refresh of the cell medium for at least 24 hours. After that, the cell medium can be changed more frequently. In another embodiment, the cell culture medium can further contain an inhibitor of cell death. For example, recently Wantanabe et al. Disclose the use of a Rho-associated kinase inhibitor, Y27632, to protect human embryonic stem cells after dissociation. Wantanabe, K. et al. , Et al., Nat. See Biotechnol, 25 (6): 681-686 (2007). This is referenced and incorporated herein by reference. In additional embodiments, the cell culture medium can contain caspase inhibitors, growth factors or other nutrients to prevent or attenuate cell death immediately after passage. Specific examples of those that can be used include HA1077, dihydrochloride, Hydroxyfasudil, Rho-kinase inhibitor, Rho-kinase inhibitor II, Rho-kinase inhibitor III, kinase inhibitor IV, and Y27632. It can be mentioned, but it is not limited to these. All of the above substances are commercially available. In yet another embodiment, the substance or element used to prevent or attenuate cell death immediately after or during cell passage can be removed from the cell medium after the cells have recovered from the passage process. .. In additional embodiments, undifferentiated embryonic stem cells can assemble effectively in standard base medium and do not require Y27632 or other involvement to maintain viability between dissociation and assembly. ..

Also, in additional embodiments, the compositions and methods of the invention can include the presence or use of surfactant compounds. In one particular embodiment, the compositions and methods of the invention comprise at least one surfactant in suspension culture. Surfactant compounds are well known in the art and are generally amphipathic. In a specific embodiment, the invention comprises the use of at least one surfactant that is anionic, cationic, nonionic or biphasic. Determining the concentration of surfactants used in the compositions and methods of the present invention is a matter of routine inspection and optimization. For example, Owen et al. Report the use of detergent compounds in cell culture methods for HeLa cells and human amniotic membrane cells. Owen et al., J. Mol. Cell. Sc, 32: 363-376 (1978). This is incorporated herein by reference. Examples of surfactant compounds that can be used include, but are not limited to, the following substances; sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfates; sodium laures sulfate (sulfate). SLES), alkylbenzene sulfonic acid ester, soap, or fatty acid salt, cetyltrimethylammonium bromine (CTAB) (hexadecyltrimethylammonium bromide), and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), Benz Alconium Chloride (BAC), Benz Etonium Chloride (BZT), Dodecyl Betaine, Dodecyl Dimethylamine Oxide, Cocamidopropyl Betaine, Palm Ammonium Foglycinate, Alkyl Poly (Ethethylene Oxide), Polyethylene Oxide and Polypropylene Oxide copolymers such as Pluronic F68, alkyl polyglycosides such as octyl glucoside, decyl maltoside, fatty alcohols, cetyl alcohols, oleyl alcohols, cocamide MEA, cocamide DEA and cocamide TEA, and / or polyoxyethylene- Solbitan monolaurate (tween) can be mentioned, but is not limited to these.

The embodiments described herein are large-scale productions that maintain the cell concentration of the system, minimize fluid shear stress, proliferate and / or differentiate hES cells by maintaining a low shear environment. Provides a way for. In particular, the present invention is low in eukaryotic cell production scale-up systems by culturing cell suspensions in 60 mm dishes, 6-well plates, rotating bottles, bioreactors (eg spinner flasks), and containers. Provide a method for maintaining a shear environment. Alternatively, a continuous perfusion system in which cells are cultured agitates or moves in a bioreactor or vessel for cell suspension, oxidation, and provision of fresh nutrients, ie, for growth and / or differentiation. I need. To obtain a cell suspension, the bioreactor vessel typically uses one or more mobile mechanical stirrers that are sources of shear stress.

It is important to establish and maintain a constant and optimized agitation shear rate to maintain cell growth and viability. For example, increased shear rates are detrimental in the following ways:
(1); Excessive shear increases energy consumption.
(2); Excessive shear interferes with diffusion on the cell membrane surface.
(3); Excessive shear may deprive a compound of bioactivity.
(4); Excessive shear deforms the cell membrane beyond the threshold tension of fracture leading to cell lysis.
Therefore, it is desirable to maintain shear within the range of 5 to 500 seconds ^-1 depending on the diameter of the cell aggregate, the sensitivity of the particular cell line to single cell dissociation and shear. Examples of shear rates in configurations useful in the methods of the invention are shown in Example 17 for aggregate diameters of 100-200 microns in diameter in 6-well dishes at rotation speeds between 60-140 rpm. .. These values estimate the time-averaged shear stresses that occur in the bulk fluid during rotation. However, the shear stress on the vessel wall is expected to be higher due to the boundary effect. Using the Lee et al. Method described above, wall shear stresses have been calculated for rotational speeds from 60 rpm to 140 rpm and are shown in Examples 17-19.

Other examples of means or devices for generating gently agitated cell suspensions are also well known to those of skill in the art. Examples include impellers such as propellers, or other mechanical means, such as bladders, fluid or gas flow based means, ultrasonic standing wave generators, platforms that sway or rotate back and forth, or Examples thereof include a combination thereof for producing a cell suspension. In the methods of the invention, the rotating platform is an exemplary means of dispersing cells within a 6-well plate, generating shear rates of less than 400 seconds- ¹ . Estimated time-averaged shear rates and shear stresses in bulk fluids are standards that all fluid mixers are associated with, regardless of the stirrer type or mechanism for producing the agitated fluid suspension. Provides a chemical element. Flow conditions within the device can vary depending on their profile and the degree of laminar or turbulent flow, and shear calculations provide the basis for equalizing the flow within the device generated by mixing by different mechanisms. provide. For example, in a 125 mL spinner flask with an impeller diameter of 4 cm, a vessel width of 6.4 cm, an impeller angle of 90 degrees, and an impeller width of 0.1 cm, in a 6-well dish with 5 mL medium rotating at l00 rpm. For a population of l00 μm in diameter, the bulk fluid produces the same time-average shear rate and shear stress.

Also, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25, The methods of the invention can be used to maintain a low shear environment for scale-up manufacturing systems for 26, 27, 28, 29, 30 days, 40 days or more, 50 days or more. Exemplary uptime is at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21, 23, 24, 25, 26, 27, 28, 29, 30 days, 40 days or more, 50 days or more.

Differentiable cells can be passaged using enzymatic or non-enzymatic methods, or by manual dissociation methods prior to and after contact with the defined medium of the invention. Non-limiting examples of enzymatic dissociation methods include the use of prostheses such as trypsin, collagenase, dyspase, ACCUTASE®. In one embodiment, ACCUTASE® is used for passage of contacted cells. When the enzymatic passage method was used, the resulting cultures could include singlets, doublets, triplets and clumps of cells, which were used. The size varies depending on the type of enzyme. A non-limiting example of a non-enzymatic dissociation method is the cell dispersal buffer. Manual passage techniques are well described in the art and are described, for example, in Schulz et al., 2004 Stem Cells, 22 (7): 1218-38. If present, the choice of extracellular matrix influences the choice of passage method, but can be readily determined by one of ordinary skill in the art.

In one specific embodiment, the method of culturing differentiable cells provides a dissociation solution of the layer of differentiable cells contained in the culture chamber prior to the dissociation breaking the cell layer into a single cell. Including doing. After dissociation, the solitary cells are placed in a stem cell culture medium and a new tissue culture chamber; where the stem cell culture medium contains a basal nutrient solution and an ErbB3 ligand. Once cultured, single stem cells are placed under conditions that allow them to grow and divide. In another specific embodiment, the method of culturing differentiable cells comprises providing a dissociation solution to the differentiable cell assembly contained in the previous culture chamber.
There, dissociation disrupts a single cell or a population of cells into a smaller population.

The disaggregation solution used in the method of the invention can be a disaggregation solution that can dissociate or dissociate cells into single cells without causing massive acute toxicity to the cells. it can. Examples of dissociation solutions include, but are not limited to, trypsin, ACCUTASE®, 0.25% trypsin / EDTA, TrypLE, or VERSENE® (EDTA) and trypsin. The method of the present invention does not require that all cells in the dense layer or suspension be dissociated into single cells, provided that at least some single cells can be dissociated and recultured.

At the beginning of culture, or after passage, cells that can differentiate at any density can be seeded and may be a single cell in the culture chamber. Various factors can regulate the cell concentration of seeded cells. For example, it includes the use of adhesive cultures, suspension cultures, specific formulations of cultured cell cultures used, growth conditions, and the expected use of the cells to be cultured. Examples of cell culture density is less like; 0.01 × 10 ⁵ cells /Ml,0.05×10 ⁵ cells /Ml,0.1×10 ⁵ cells /Ml,0.5×10 ⁵ cells / ml, 1.0 × 10 ⁵ cells /Ml,1.2×10 ⁵ cells /Ml,1.4×10 ⁵ cells /Ml,1.6×10 ⁵ cells /Ml,1.8×10 ⁵ cells / ml, 2.0 × 10 ⁵ cells /Ml,3.0×10 ⁵ cells /Ml,4.0×10 ⁵ cells /Ml,5.0×10 ⁵ cells /Ml,6.0×10 ⁵ cells / ml, 7.0 × 10 ⁵ cells /Ml,8.0×10 ⁵ cells /Ml,9.0×10 ⁵ cells / ml or 10.0 × 10 ⁵ cells / ml, or more, for example 5 × up to 10 ⁷ cells / mL are cultured shows the survival of good cell. Any value between these values can be adopted.

In addition to the above, as used herein, the term "cell concentration manipulation", "manipulated cell concentration" or an equivalent expression thereof, is the manipulation of cell density in a manufacturing process or system for proliferation. Producing sex or differentiated hES cell cultures. Such cell concentrations are such that nutrients such as vitamins, minerals, amino acids, biotransformers, etc. supplied to the system, or environmental conditions such as partial pressure of oxygen are sufficient to maintain cell vigor. To say. Alternatively, such cell concentration refers to those under which waste can be removed from the system at such a rate that cell vigor can be maintained. Those skilled in the art can easily determine such cell concentrations.

Working cell concentration can be maintained is at least about 0.5 × 10 ⁶ cells / mL. Typical scale-up system actuated cell concentrations range from about 0.5 × 10 ⁶ cells / mL to about 25 × 10 ⁶ cells / mL. Exemplary concentrations range from about 2.5 × 10 ⁶ cells / ml, 22 × 10 ⁶ cells / mL to a maximum of 5 × 10 ⁷ cells / mL. In the methods of the invention, cell viability is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and about. Up to 100%. Other scale-up system working cell concentrations and acceptable cell viability levels will be determined by techniques recognized by those of skill in the art and well known to those of skill in the art. For example, batch, fed-batch
And in a continuously fed configuration, the cell concentration may be between about 0.5 × 10 ⁶ cells / mL and 15 × 10 ⁶ cells / mL.

Also, differentiateable cells are available for screens for molecules or elements that affect their formability or other properties. For example, differentiateable cells can be used to identify agents that cause apoptosis, differentiation, or differentiated linenage originating from proliferative or differentiateable cells.

Differentiable cells are successfully cultured in, for example, 96-well plates or 384-well plates, but not limited to high-volume treatment settings, as the compositions and methods of the invention allow single cell passage. It was. FIG. 16 shows morphology and alkaline phosphatase staining of BG02 cells cultured in DC-HAIF on both 96-well and 384-well plates using the methods of the invention. Briefly, hESCs cells passaged using ACCUTASE® and plated on 96-well and 384-well plates formed colonies similar to those observed using other cultures. The rate was shown. In addition, the cells colonized and successfully expanded in a smaller environment for 5 days. These smaller cultures remained morphologically undifferentiated and were uniformly positively stained with alkaline phosphatase, a marker for undifferentiated cells. In addition, ACEA Biosciences, Inc. A 96-well culture apparatus that provides real-time measurements of impedance that can measure cell proliferation and viability by using the RT-CES® method from (www.aceavio.com). I was able to grow hESCs (not shown). Such an approach would allow identification and quantification of subtle or immediate effects in differentiated cells without the use of labels. It will also enable real-time measurement of proliferation, apoptosis and morphology changes.

The compositions and methods of the invention may actually include any combination of line segments described above or elsewhere herein. However, the compositions and methods of the present invention include basal nutrient solutions and means of stimulating ErbB2-derived tyrosine kinase activity. Those skilled in the art will appreciate that the design of the protocol will result in different components of the compositions and methods of the invention. Thus, one embodiment of the invention relates to culturing differentiateable cells in 96-well plates and / or 384-well plates. Using the compositions and methods of the present invention, cell culture chambers, i.e. cultures, are not limited to a particular size. Therefore, the methods described herein are by no means limited by specific culture chamber dimensions and / or means and devices for generating cell aggregates.

The compositions and methods described herein have some useful features. For example, the compositions and methods described herein are useful for modeling the early stages of human development. In addition, the compositions and methods described herein are useful for therapeutic interventions in diseases and are due to the development of pure tissue or cell types, such as disorders of neurodegeneration, diabetes or renal failure. It is useful for.

The cell types that differentiate from differentiateable cells are not limited to these, but much of the research and development, including the production of cells for drug discovery, drug development and testing, toxicity, therapeutic purposes and basic science research. Has applications in the field of. These cell types express molecules of interest in a variety of research areas. These include molecules known to be required for the function of the various cell types described in the standard reference text. These molecules include, but are not limited to, cytokines, growth factors, cytokine receptors, extracellular matrix, transcription factors, secreted polypeptides and other molecules, and growth factor receptors.

It is contemplated that the differentiateable cells of the present invention can differentiate through contact with the cell differentiation environment. As used herein, the term "cell differentiation environment" refers to cells that are induced to differentiate into differentiateable cells or to enrich human cell culture within differentiated cells. The culture conditions are shown. Desirably, the differentiated cell lineage caused by growth factors will be homogeneous. The term "uniformity" is about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. , 95%, 96%, 97%, 98%, or 99% or more of the population containing the desired cell lineage.

A cell differentiation medium or environment can be used to partially or reversibly differentiate the differentiateable cells of the invention. According to the present invention, the medium of the cell differentiation environment has various components, for example, KODMEM medium (Knockout Dulbecco's Modified Eagle's Medium), DME.
F12 medium of M, Ham, FBS (fetal bovine serum), FGF2 (fibroblast growth factor 2), KSR or hLIF (human leukemia inhibitory factor) can be included. In addition, the cell differentiation environment can include supplements such as L-glutamine, NEAA (non-essential amino acid), P / S (penicillin / streptomycin), N2, B27 and β-mercaptoethanol (βME). It is contemplated that additional elements such as the following can be added to the cell differentiation environment; fibronectin, laminin, heparin, heparin sulfate, retinoic acid, members of the epidermal growth factor family (EGFs); FGF2, FGF7, FGF8, And / or members of the fibroblast growth factor family (FGFs), including FGF10, members of the platelet-derived growth factor family (PDGFs); conversion growth factor (TGF) / bone morphogenetic protein (BMP) / growth and differentiation factors ( GDF) family of antagonists, such as, but not limited to, nogin, holistatin, cordin, gremlin, cerberus / DAN family proteins, bentopin, high dose actibine, and amnionless; variants or their functional Fragment. TGF / BMP / GDF antagonists can also be added in the form of TGF / BMP / GDF receptor-Fc chimeras. Other factors that can be added include, but are not limited to, molecules capable of activating or inactivating signaling through the Notch receptor family, for example, delta-like (. It contains Delta-like and Jagged family of proteins, as well as Notch processing or cleavage inhibitors, or variants and functional fragments thereof. Other growth factors include insulin-like growth factor family (IGF), insulin, wingless related (WNT) factor family, and hedgehog. Factor family), variants or functional fragments thereof. To promote proliferation and survival of endoderm stem / progenitor cells, endoderm stem / progenitor cells, germ layer stem / progenitor cells, or final endoderm stem / progenitor cells, and the origin of these progenitor cells Additional elements can be added to promote the survival and differentiation of the body.

The compositions described herein are useful for screening test compounds to determine if the test compounds regulate pluripotency, proliferation, and / or differentiation of differentiateable cells. One of skill in the art can ascertain the pluripotency, proliferation, and / or differentiation of easily differentiateable cells. Non-limiting methods include measurement of cell morphology, expression of various markers, teratoma formation, cell number, and impedance.

Progression of differentiateable cells into the desired cell linenage, or maintenance in an undifferentiated state, quantifies the expression of marker gene characteristics of the desired cell linenage and the deficiency of marker gene characteristics of the differentiateable cell type. Can be monitored by. One way to quantify the gene representation of such marker genes is the use of quantitative PCR (Q-PCR). Methods of performing Q-PCR are well known in the art. Other methods known in the art can also be used to quantify marker gene expression. Marker gene expression can be detected by using an antibody specific for the labeled gene of interest.

In certain embodiments, screening methods include methods that identify compounds that can regulate pluripotency, proliferation and / or differentiation of differentiateable cells, including:
(A); To provide differentiateable cells;
(B); culturing cells in a composition containing a basal nutrient solution and ErbB3 ligand and virtually free of serum;
(C); Contacting cells with the test compound; and determining whether pluripotency, proliferation, and / or increase or decrease in differentiation occurs in cells contacted with the compound.
If the above-mentioned increase occurs here, it indicates that the compound regulates pluripotency, proliferation, and / or differentiation. In certain embodiments, the ErbB3 ligand is HRG-β. In other embodiments, the ErbB3 ligand can be substituted with the test compound to determine the effect of the test compound. For example, the pluripotency, proliferation, and / or effect on differentiation that occurs with a test compound is compared to the pluripotency, proliferation, and / or effect on differentiation that occurs with an ErbB3 ligand, and the test compound on differentiateable cells. The effect of can be determined. It is assumed that all the compositions described herein can be used in the screening method of the present invention.

In yet another embodiment, the ErbB3 ligand (ErbB2-derived tyrosine kinase (t).
The effect of yrosine kinase) activity) on cells in the absence of deficiency of the ErbB3 ligand (ErbB2-derived tyrosine kinase activity) can be determined.

The methods described herein can be used to produce compositions that are substantially free of other cell types, including the desired cell linenage. Alternatively, a composition comprising a mixture of differentiateable cells and the desired cell lineage can be prepared.

In some embodiments of the invention, cells of the desired cell linenage can be isolated by using such cell-specific affinity tags. One example of a target cell-specific affinity tag is present on the cell surface of a target cell found in cell cultures produced by the methods described herein, but is substantial for other cell types. An antibody that is specific for a marker polypeptide that is not present in the art.

The present invention also relates to a kit, which comprises at least one compound capable of stimulating ErbB2-derived tyrosine kinase activity and a basal nutrient solution. In one embodiment, the kit comprises at least one ErbB3 ligand as described herein. In another embodiment, the kit contains more than one ErbB3 ligand. In another embodiment, the kit comprises at least one TGF-β, TGF-β family, variants or functional fragments thereof, as described herein. In yet another embodiment, the kit comprises more than one TGF-β, TGF-β family, variants or functional fragments thereof. In yet another embodiment, the kit comprises at least one fibroblast growth factor, variants or functional fragments thereof. In another embodiment, the kit comprises two or more fibroblast growth factors, variants or functional fragments thereof. In a specific embodiment, the kit comprises at least one of FGF-7, FGF-8, FGF-10, FGF-22, a variant thereof or a functional fragment thereof. In another embodiment, the kit comprises serum albumin. In yet another embodiment, the kit comprises serum, at least one insoluble substrate described herein, and / or at least one dissociation solution.

The kits of the invention may also include all combinations of ingredients described above or anywhere herein. As will be appreciated by those skilled in the art, the ingredients supplied to the kit of the present invention will vary depending on the application for the kit. Therefore, kits can be designed to perform the various functions described in this application, and the components of such kits will therefore be different.

Various documents are referenced throughout this application. The disclosures of these publications are incorporated herein by reference in their entirety in order to more fully describe the state of the art in the art of the present invention.

Examples Human embryonic stem cell line BG01v (BresaGen, Inc., Asains, Georgia) was used in some of the experiments described herein. The BG01v hESC line is a karyotype mutant cell line. The cell line shows a stable karyotype containing specific three chromosomes (karyotype: 49, XXY, +12, +17). Parental cultures were maintained as described above (Schulz et al., 2003, BMC Neurosci., 4:27; Schulz et al., 2004, Stem Cells, 22 (7): 1218-38; Rosie et al., 2004, Dev. Dynamics, 229: 259-274; Brimble et al., 2004 Stem Cells Dev., 13: 585-596). Briefly, cells are cells coated with MATRIGEL® or fibronectin, DMEM: F12 and 20% KSR, 8 ng / mL FGF2, 2 mM L-glutamine, 1x non-essential amino acids, 0.5 U / mL. Collagenase in conditioned medium (Sigma, St. Louis, Missouri, USA) from mouse fetal fibroblasts (MEFs) (MEF-CM) containing penicillin, 0.5 U / mL streptomycin, 0.1 mM β-mercaptoethanol. It was grown by succession.

The defined culture (DC) media tested herein are DMEM / F12, 2 mM Glutamax, Ix non-essential amino acids, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, 10 microg. / ML iron-binding globulin (all from Invitrogen, Carlsbad, CA, USA), 0.1 mM β-mercaptoethanol (Sigma), 0.2% fatty acid-free Corns fraction (Cohn's fractions) V BSA (Serologicals) , 1x Trace Element mixes A, B, and C (Cellgro), and 50 microg / mL ascorbic acid (sigma). Variable levels of recombinant growth factor were used. The factors include FGF2 (sigma), LongR3-IGFl (JRH Biosciences), Heregulin-β EGF domain (HRGβ, Peprotech), TGFβ (R & D systems),
Examples include nodal (R & D systems), LIF (R & D systems), EGF (R & D systems), TGFα (R & D systems), HRGα (R & D systems), BMP4 (R & D systems), and activin A (R & D systems). LongR3-IGFl is a modified version of IGFl with reduced affinity for IGFl-binding proteins. Some of them are expressed in hESCs. DC-HAIF is the previously defined culture medium containing 10 ng / mL HRG-β, 10 ng / mL activin A, 200 ng / mL LR-IGFl, and 8 ng / mL FGF2. DC-HAI is a previously defined culture medium comprising 10 ng / mL HRG-β, 10 ng / mL activin A, and 200 ng / mL LR-IGFl. The DC-HAIF and DC-HAI, LR-IGFl components are, of course, replaced by IFGl.

Plates coated with MATRIGEL® are grown factor reduced BD MATRIGEL® Matrix (BD Biosciences, Franklin Lakes)
, New Jersey, USA) is prepared by diluting in cold DMEM / F-12 to a final concentration range from about 1:30 to about 1: 1000. In one embodiment, the concentration of MATRIGEL® is about 1: 200. A 1 ml / 35 mm dish was used to coat the dish at room temperature for 1-2 hours, or at 4 ° C. for at least overnight. Cultures were stored at 4 ° C. for up to 1 week. Immediately before use, the MATRIGEL® solution was removed. For the conditions tested, parent cultures were plated in 6-well dishes for comparison of multiple conditions. Cultures were typically plated directly under test conditions. Cultures were evaluated daily starting 4-5 days after plating and graded based on morphological criteria. Rating scales 1-5 are to examine the entire culture to determine the overall proportion of undifferentiated colonies, their relative size, the proportion of colonies showing overt differentiation or the portion of corny It was good. Grade 5 exhibits an "ideal" culture with large undifferentiated colonies and negligible differentiation. Grade 4 shows a very good culture, but shows some overt differentiation. Grade 3 shows acceptable culture, but approximately half of the colonies show overt differentiation. Grade 2 is predominantly differentiated and there are occasional putative undifferentiated cells. Grade 1 cultures contained differentiated colonies, or the cultures did not adhere or survive. Cultures that showed good expansion of undifferentiated cells were passaged to evaluate longer-term cultures under these conditions.

Expression of ErbBl-3, Nrgl, and ADAM19 in Example 1-BG01v cells

Real-time RT PCR was used to show the expression of ErbBl-3, neuregulin, and ADAM-19 in BG01v cells (Fig. 1). 100 ng / mL L
BG01v cells cultured in DC medium as described above, containing ongR3-IGFl (LR-IGFl), 8 ng / mL FGF2, 1 ng / mL Actibin A, are harvested and RNA is harvested according to the manufacturer's instructions. Prepared using the RN easy mini kit (Qiagen). The first strand cDNA was prepared using the iScript kit (Biorad). Real-time PCR was then performed using the MJ Research Opticon thermal cycler.

Takuman (TaqMan) for ADAM19 (Hs00224960_ml), EGFR (Hs00l93306_ml), ErbB2 (Hs001704033_ml), ErbB3 (Hs00176538_ml), NRGl (Hs00247620_ml), OCT4 (Hs007428960_ml), OCT4 (Hs00742896_ml) GAPDH was used in the TaqMan universal PCR (Applied Biosystems). A real-time amplification plot showing the expression of these transcriptions in undifferentiated BG01v cells is shown in FIG.

Example 2-Suppression of ErbB2 slows the proliferation of BG01v cells

The EGF domain family of ligands binds to the ErbB family of receptor tyrosine kinases. To examine the known inhibitory effect of ErbB tyrosine kinases in hESCs, BG01v cells were placed in a 6-well tray on MATRIGEL® diluted 1000: 1 at 100 ng / mL LongR3-IGFl, 8 ng / mL FGF2. , And 1 ng / mL Actibin A were plated in a defined culture medium (DC) containing Actibin A. The next day, DMSO (Carrier Control), 50 nM-20 micro M AG1478 (ErbBl inhibitor), or 100 nM-20 micro M AG879 (ErbB2 inhibitor) was added with fresh medium. The cells were cultured for 5 days with daily medium changes. Cultures were fixed and stained for alkaline phosphatase activity.

Subconfluent colonies of AP + BG01v cells were observed in controls and AG1478 cultured cells (FIGS. 2A and 2B). Neither DMSO nor AGl478 (50 nM-20 microM) has been shown to have a clear effect on cell proliferation. AG879, however, substantially banned cell growth at 5 microM (Fig. 2C) and caused cell death at 20 microM (not shown). Cultures grown on AG879 did not show differentiation and appeared to maintain pluripotent morphology and alkaline phosphatase activity. This indicates that AG879 suppresses proliferation without causing differentiation, suggesting that BG01v cells rely on ErbB2 signaling for cell survival. Conversely, BG01v cells grown under the same conditions as above do not appear to rely on the ErbBl signal for proliferation.

Example 3-BG01v cells are maintained in a defined medium containing heregulin

Expression of ErbB2 and ErbB3 and inhibition of proliferation at AG879 suggested that BG01v cells were active in endogenous ErbB signaling and that they were able to respond to exogenous HRG-β. BG01v cells grow in DC medium containing 10 ng / mL HRG-β, 200 ng / mL LongR3-IGFl, 8 ng / mL FGF2, and 10 ng / mL activin A on MATRIGEL® diluted 1: 1000. (FIGS. 3A and B). Cells grew longer than 4 or 20 days, showed undifferentiated morphology, and did not show high idiopathic differentiation.

In addition, BG01v cells were maintained in DC media containing 10 ng / mL HRGβ, 200 ng / mL LongR3-IGFl, and 10 ng / mL activin A for 2 generations, or longer than 13 days. These cultures grew normally and showed very low idiopathic differentiation. This indicates that BG01v cells could be maintained under defined conditions in the presence of HRGβ in the absence of FGF2.

Example 4-Role of ErbB2-derived tyrosine kinase in ES cells

RT-PCR showed that mESCs expressed ADAM19, neuregulin l (Nrgl), and ErbBl-4 (Fig. 4A). In mESCs, ErbB2 and 3 were shown to be expressed at higher levels than ErbB1, and lower levels of ErbB4 were detected. These data suggest that endogenous HRG-β could be involved in promoting mESC self-renewal.

Expression of ErbB receptor transcripts in mouse fetal fibroblasts (MEFs) was also examined (FIG. 4B). MEFs are a heterogeneous population of cells derived from the viscera of E12.5-13.5, which has historically been used to maintain mouse and human EC cells and embryonic stem cells. Expression of Nrgl and Adam19 in this population suggests that the HRG-β ect domain is also present in MEF-conditioned medium and can exert a significant effect on pluripotency.

AG1478 and AG879 were used to investigate the role of HRG / ErbB signaling in mouse embryonic stem cells. Rl mouse ES cells are in the standard state of DMEM, 10% FBS, 10% KSR, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, IxNEAA, 1 mM sodium pyruvate, 1000 U / mL LIF (ESGRO), 0.1 mM. It was maintained at βME and passaged with 0.5% trypsin / EDTA. The 2 × l0 ⁵ cells / well 1: is plated in 6-well in a tray on a diluted MATRIGEL (TM) 1000. The day after plating, DMSO (Carrier Control), 1-50 microM AG1478, or 1-50 micron M AG879 was added with fresh medium. The cells were cultured for an additional 8 days, changing medium daily. Cultures were then stained for immobilized alkaline phosphatase activity.

DMSO and 1-50 micro-M AGl478 had no apparent effect on cell proliferation. "Sub-confluent" colonies of alkaline phosphatase-positive mESCs were observed (Fig. 5A-C). However, AG879 may have substantially banned cell growth at 50 micrometers (comparison of FIGS. 5D and 5F) and slowed proliferation at 20 micrometers (FIG. 5E). The mESCs grown on AG879 showed no differentiation and maintained pluripotency morphology and alkaline phosphatase activity.

The results suggested that AG879 suppressed proliferation and did not show differentiation of mESCs, suggesting that mESCs require ErbB2 signaling for proliferation. Conversely, mESCs do not appear to rely on ErbBl signaling for proliferation. The concentration of AG879 required to suppress proliferation was about 10-fold higher for mESCs than for BG01v cells grown under defined conditions. Serum used in mESC conditions interfered with drug activity, AG879 has a lower affinity for mouse ErbB2 tyrosine kinase than human ErbB2 tyrosine kinase, or ErbB2 plays a slightly different role in different species of embryonic stem cells. It shows whether it can be fulfilled.

Another highly selective inhibitor of ErbB2 tyrosine kinase, tyrphostin AG825 (Mullillo et al. 2001 Cancer Res 61, 7408-7421), was used to investigate the role of ErbB2 in human ESCs. AG825 markedly prohibited the growth of hESCs growing in conditioned medium (CM) (FIG. 6A). AG825 suppressed proliferation without causing extensive cell death and was able to maintain the survival of hESCs for more than 5 days (not shown). Western blotting showed that AG825 prohibited the automatic phosphorylation of ErbB2 with tyrosine-1248 in starved / heregulin (HRG) pulsed hESCs growing on DC-HAIF. (Fig. 6B). Therefore, signaling of ErbB2 division severely suppressed hESC proliferation. To establish hESCs under defined growth conditions, cultures were directly passaged from CM conditions to DC-HAIF, exhibiting minimal idiopathic differentiation (FIG. 6C). The colony and cell metrology assay method confirmed that LongR3-IGFl and HRG play a major role in self-renewal and proliferation in one context of embodiments of the invention (FIGS. 6D and 6E). Phosphorylation of IGFlR, IR, FGF2α, ErbB2, and ErbB3 was observed in both steady-state DC-HAIF cultures and DC-HAIF starved cultures that were pulsed with DC-HAIF (FIG. 6F). ..

Example 5, Culture of Mouse ES Cells Under Defined Conditions

To further investigate the role of HRG / ErbB2 signaling in mouse embryonic stem cells, Rl ES cell proliferation was examined in DC medium using a combination of growth factors. 1 × 10 ⁵ cells / well were plated in a 6-well tray, coated with 0.2% gelatin, 10 ng / mL HRG-β, 100 ng / mL LongR3-IGFl, 1 ng / mL Actibin A, 1000 U. / ML mouse LIF or 10 ng / mL
Incubated in DC containing a combination of BMP4 (Table 1 below). Proliferation was observed for 8 days.

Viable colonies grew only under conditions containing at least LIF / HRG-β or LIF / BMP4 (Table 1). No clear additional benefit was observed when LongR3-IGFl or activin was added to these combinations. Normal growth was observed in control parent cultures. And viable colonies were not observed in the defined medium without growth factors. See Table 1.

Quantitative measures are performed on a selected combination of 1: 1000 MATRIGEL® in a 6-well tray and 10 or 50 ng / mL HRG-beta, EGF, 1000 U / mL LIF or 10 ng / mL BMP4. × a l0 ⁵ cells / well it was performed by plating. The culture was grown and fixed for 8 days. Then, the number of alkaline phosphatase colonies was counted (Fig. 7A). No colonies were observed without growth factors under the defined conditions. And less than 45 colonies were observed with the combination of HRG-β, HRG-β / EGF, and HRG-β / BMP. 1358 colonies were observed only with LIF, while 4114 and 3734 colonies were observed with a combination of 10 ng / mL HRG-β / LIF and 50 ng / mL HRG-β / LIF, respectively. This showed that under the defined conditions, only LIF provided a substantial pluripotency signal. HRG-β showed a large synergistic effect with LIF, and in this analytical evaluation, the number of proliferative mESC colonies was more than doubled. In addition, the low-magnification image of this analytical evaluation shows this synergistic growth promoting effect (FIG. 7BG).

Characterization of pluripotency of human embryonic stem cells (hESCs) maintained in Example 6-DC-HAIF Multiple approaches have been used to confirm the maintenance of hESCs formation in DC-HAIF. BG02 cells cultured in DC-HAIF for 6 months (25s) maintained the potential to form complex teratomas (Fig. 8A) and were representative of the three in vitro germ layer layers. Transcriptional analysis was used to compare global expression in hESCs cells maintained in CM and DC-HAIF (Liu et al., 2006, BMC Dev Biol 6, 20). More than 11,600 transcriptions were detected in 10-32 passage BG02 cells grown in DC-HAIF and in 64 passage BG02 cells grown in CM. Transcription of about 10364 was common in all populations, including known hESC markers such as CD9, DNMT3, NANOG, OCT4, TERT and UTFl (not shown) (FIG. 8C). High correlation coefficients were observed in the comparison of CM and DC-HAIF cultures (R ² select = 0.928) and in early and late passage cells (R ² select = 0.959) (Figure). 8D). Hierarchical cluster analysis showed that BG02 cells maintained in DC-HAIF were tightly retained and had similar similarities to BG02 and BG03 cells maintained in CM (FIG. 8E). These data are consistent with previous analysis, indicating that undifferentiated hESCs are tightly clustered compared to embryoid bodies or fibroblasts (Liu et al., 2006, BMC Dev Biol 6, 20). ing. Therefore, cells maintained with the compositions of the present invention can maintain key markers of pluripotency. Therefore, the compositions of the invention can be used as a simple medium to support the self-renewal of differentiateable cells.

Maintenance of human embryonic stem cells (hESCs) in humanized extracellular matrix (ECMs) in Example 7-DC-HAIF

To investigate the role of ErbB2 signaling and develop defined media for hESCs, DC-HAIF cultures were coated with growth factor-reduced l: 30 MATRIGEL®. Initially spread on top. It was successfully maintained in the long term on a substrate diluted 1: 200 or 1: 1000 (Fig. 9A). Also, on a tissue culture dish coated with human serum (Fig. 9B); human fibronectin (Fig. 9C); or VITROGRO® (Fig. 9D), which is a proprietary humanized ECM (ECM). BG02 and CyT49 hESCs could be maintained longer than 5 passages.

Example 8-Single cell passage of human embryonic stem cells (hESCs)

Several research groups have shown that certain triploids, markedly hChrl2 and 17, are accumulated in hESCs under certain partial optimal culture conditions (Baker et al., 2007 Nat. Biotech. 25 (2): 207-215). The triploid appearance appears to be most directly associated with inadequate cell survival when the culture is separated into single cells during passage, and these aneuploid cell harboring. The presumed strong selective growth benefit is provided to the cells. Conversely, hESCs growing on DC-HAIF in one embodiment of the invention maintained high viability by plating after isolation into single cells (FIGS. 10A-D). BG0l and BG02 cells were maintained in normal karyotype after passages> 18 and 19 respectively under the ACCUTASE: Registered Trademark (Fig. 10E). Maintenance of normal karyotype in cells showed that dissociation of hESC cultures into single cells did not lead to these trichromosomal accumulations of hESCs that were originally maintained in DC-HAIF. BG0l and BG02 cultures are also passaged by dissociation into single cells using multiple passage reagents (FIG. 11). Medium was split and karyotyped with ACCUTASE®, 0.25% Trypsin / EDTA, TrypLE, or Verseen® for 5 passages. The data show that culture and passage of hESCs in the compositions of the present invention maintained normal karyotype in at least two human embryonic cell lines using a variety of cell dissociation reagents.

Large-scale expansion of undifferentiated hESCs is also possible using the compositions of the present invention. The first confluent culture of BG02 cells in 60mm plates (confluent culture) is a single experiment for 20 days in a DC-HAIF, 1.12 × ^{l0 10}
It was propagated to more cells in 4 passages. The culture remained undifferentiated and, according to fluid cell assay, more than 85% of the cells in the batch expressed pluripotent markers such as OCT4, CD9, SSEA-4, TRA-1-81. Was maintained (Fig. 12A). Expression of other pluripotent markers was also observed by RT PCR analysis. On the other hand, markers of differentiated linenage α-fetoprotein, MSXl, and HANDl were not detected (Fig. 12B). Fluorescence in situ hybridization analysis revealed that the cells cultured and subcultured in DC-HAIF were hChrl2 (98% 2-copy), hChrl7 (98%, 2-copy), hChrX (95%, 1-copy). And for hChrY (98%, 1-copy), the expected copy number was maintained (FIG. 12C). Karyotype analysis also showed that normal ploidy chromosomal content and banding profile were maintained in these cells.

Example 9-Different effects of insulin and IGFl on hESCs when applied at physiological concentrations

Essentially all of the previously reported culture conditions for hESCs contain physiologically excessive amounts of insulin capable of stimulating both IR and IGFlR. To distinguish the activity of insulin and insulin substitutes as compared to IGFl, hESCs are cultured in defined media conditions with physiological concentrations of these growth factors. Insulin and IGFl concentrations range from about 0.2 to about 200 ng / mL and cell proliferation is monitored by counting cells after 5 days. The medium that spreads successfully is passaged 5 times in a row. Physiological concentrations of insulin do not support expansion of hESC cultures, whereas physiological concentrations of IGFl support expansion of hESC cultures. This indicates that the activity of insulin or insulin substitute cannot replace IGFl, and IGFl and insulin (or insulin substitute) exert different classes of biological activity with respect to their effects on hESCs.

Example 10-Method of screening the effects of supplements

To initially assess the effect of vitamins B ₁₂ and B _{6 on} the growth or differentiation of hESCs growing at intermediate densities, BG02 cells were used in ACCUTASE® at 1 × 10 ⁵ cells / well. , 6 Well trays were plated and divided in the defined culture (DC) medium. DC medium is 10 ng / mL HRG-β, 200 ng / mL
It contains LongR3-IGFl and 10 ng / mL FGF10. Vitamin B ₆ (0.5 microM) and / or vitamin B ₁₂ (0.5 microM) is added to the test wells. The number of cells under each condition is counted after 7 days. The number of cells and colonies in both experimental and control wells provides a prospect of the effect of vitamins B ₁₂ and B ₆ on cell growth.

In addition, differentiation markers such as OCT4 can be evaluated in test wells to determine the effect of additives and supplements on the differentiation status of differentiateable cells.

Medium of hESCs in the absence of Example 11-FGF2

BG02 cells were maintained in DC-HAI for a long period of time (20 passages) (Fig. 13A). BG0l cells were also continuously passaged within DC-HAI. Both were in the absence of FGF2. The culture showed normal expansion of undifferentiated colonies over the entire duration of the culture, with no deterioration or no overt differentiation. Maintenance of normal male karyotype in BG02 cultures was shown after 6 passages within DC-HAI (FIG. 13B, 20/20 mid-term spread of normal mitosis).

Transcriptional analysis was used to compare global expression in hESCs cells maintained with DC-HAIF and DC-HAI. Total cell RNA was isolated from hESCs using Trizol (Invitrogen) and treated with DNase I (Invitrogen) according to the manufacturer's suggested design of experiments. Sample amplification was performed with 100 ng of total RNA using the Illumina RNA Amplification kit. And labeling is biotin-in a 1: 1 ratio with unlabeled UTP.
It was achieved by adding 16-UTP (Perkin Elmer Life and Analytical Sciences). Labeled amplification products (700 ng per array) are Illumina Sentrix Human-6 Expression Beadchips containing 47,296 transfer probes according to the manufacturer's instructions (Illumina, Inc.). ) And hybridized. The array is scanned with an Illumina Bead Array Reader confocal scanner for primary data processing, background removal, and data analysis using Illumina Bead Studio software as directed by the manufacturer. It has been executed. A minimum detection confidence score of 0.99 (calculated cutoff indicated that the target sequence signal was distinguishable from the negative control) was used to distinguish the presence or absence of transcripts. .. Data analysis was performed using the parallel approach described for other hESC samples (Liu et al. 2006, BMC Dev Biol 6:20). Hierarchical clustering is performed as described above (Liu et al., 2005, BMC Dev Biol 6:20) and is a similarity metric that uses the differentiated expressed genes identified by ANOVA. Based on mean concatenation and Euclidean distance (p <0.05). Details of the sensitivity and quality control tests used in array manufacturing, as well as a detailed description of the algorithms used in the bead studio software, are available from Illumina Inc (San Diego, California). The majority of the detected transcriptions were expressed in both DC-HAIF and DC-HAI BG02 cultures and contained known hESC markers such as CD9, DNMT3, NANOG, OCT4, TERT and UTFl (not shown). A high correlation coefficient was observed in the comparison of DC-HAIF and DC-HAI cultures (R2select = 0.961) (FIG. 14). Hierarchical clustering analysis shows that BG02 cells maintained within DC-HAI are grouped tightly and DC-HAI as well as multiple culture types of BG02 and other hESC lines.
It possessed similarities to cells maintained in F (Fig. 15). These data are consistent with previous analyzes showing that undifferentiated hESCs were clustered tightly compared to embryoid bodies or fibroblasts. (Liu et al., 2006, BMC Dev Biol 6:20)

In addition, BG02 cells maintained within DC-HAI differentiated into representative species of endoderm, ectoderm and mesoderm in complex teratomas formed in SCID beige mice (not shown) and exogenous FGF2. We showed the maintenance of pluripotency in cultures grown in the absence of.

To determine if exogenous FGF2 was required for single cell passage, BG0l cells were passaged with ACCUTASE® and only 10 ng / mL HRG-β and 200 ng / mL LongR3-IGFl (DC-HI). It was grown under defined conditions including. These DC-HI cultures were maintained for 10 passages and did not show a clear slowdown of differentiation or proliferation.

These studies clearly showed that exogenous FGF2 was not required when maintained in a defined medium containing hESCs minimal containing heregulin and IGFl. In addition, FGF2-free cultures possessed the basic properties of pluripotency, including in vivo differentiation and transcriptional profiles into mesoderm, endoderm, and ectoderm.

Example 12-Suspension culture

The starting medium for BG02 cells was maintained in DC-HAIF medium on a dish coated with 1: 200 matrigel, passaged and divided with ACCUTASE®, as described herein. It was. To initiate suspension culture, BG02 cells were disintegrated with ACCUTASE® and 1.6, 3 in DC-HAIF medium.
, Or 6 × l0 ⁵ cells / mL were placed in low attachment 6-well tray at a density of (in 3mL volume, 0.5, 1, or 2 × l0 ⁶ cells). The tray was placed on a rotating platform at 80-100 rpm in a humidified incubator with 5% CO ₂ . Under these conditions, hESCs were morphologically fused to viable globules within 24 hours.

The well medium was changed on the second day and daily thereafter. The suspended population continued to grow and grew over time without overt signs of differentiation (Fig. 17). Some of the spheres remained aggregated during the process of culturing, but some populations became much larger than most. In addition, non-spherical aggregates were observed during the coalescence process during the first few days of culture. For this continuous assembly only, 38 microg / mL DNase I was included in the first 24 hours of suspension culture. However, this approach appeared to be useful for relatively large early populations, as smaller populations are formed in the presence of DNase I. However, it is unclear whether exposure to DNase I will consistently harden these populations and destroy them during separation, reducing sphere coalescence followed by DNase I treatment.

Suspension cultures were dissociated with ACCUTASE® approximately every 7 days to produce new spheres. Although densities varied from experiment to experiment, spheres made at densities within this range (1.6-6 x l05 cells / milliliter) were morphologically differentiated for longer than 12 passages or longer than 80 days. It could be maintained in the medium without any signs. FISH analysis of continuously passaged suspended hESCs was performed to determine the number of common aneuploid chromosomes. BG02 cells grown in suspension for 6 passages were hChr12 (96%, 2-copy, n = 788), hChr17 (97%, 2-copy, n = 587), hChr X (97%). Normal counts were shown for 1, copy, n = 724), and hChr Y (98%, 1 copy, n = 689).

Example 13-Expansion of differentiateable cells in suspension culture

Unlike embryoid body cultures in the presence of serum or differentiation inducers, the suspended population of hESCs in DC-HAIF did not appear to differentiate. No overt organ endoderm was observed, and there was no formation of proamniotic cavities-like structures or classical signs of embryoid body differentiation. To more closely examine the lack of differentiation, the cultures were plated back to adhesion conditions on MATRIGEL® diluted 1: 200 and cultured in DC-HAIF. These cultures were essentially undifferentiated and did not show overt morphological signs of increasing differentiation, such as the presence of larger, flattened cells, or structural regions.

Cell counts were used to assess the relative growth rate of cells in suspension cultures compared to adherent cultures. In this experiment, the adherent culture of BG02 cells was subcultured with ACCUTASE® and approximately 1 × 10 ⁶ cells were placed in suspension or adherent culture wells. Individual wells were counted and plotted on a logarithmic scale for 1-6 days (Fig. 18). During adherent culture, they showed a higher initial percentage of hESCs after 24 hours (about 90% vs. about 14%), but the growth rate was subsequently comparable. This showed that hESCs could grow almost as rapidly in traditional adherent and suspension cultures. Cell counts made during passage allow the amount of expansion possible in this simple suspension system to be measured. In some cultures, in seeding 5 × 10 ⁵ cells after 7 days to about 10 ⁷ or more cells occurs. The expansion after 7 days in suspension culture was equal to about 20-fold or higher expansion, with the largest observed expansion being about 24-fold.

Example 14-Characteristics of differentiated cells expanded in suspension culture

Quantitative RT-PCR (qPCR) was used to compare gene expression of hESCs grown in suspension and adhesion cultures in DC-HAIF. Similar levels of OCT4, a marker for pluripotent cells, were observed in both culture types, confirming that the cultures maintained in suspension cultures were essentially undifferentiated. The final endoderm marker, SOX17, was not expressed in either population of hESCs. In addition, qPCR analysis examined the possibility that suspension-cultured hESCs differentiate into the final endoderm as a population in suspension. Adhesive and suspended hESCs were differentiated using the same conditions. The hESC cultures were treated with RPMI containing 2% BSA, 100 ng / mL Actibin A, 8 ng / mL FGF2, and 25 ng / mL Wnt3A for 24 hours and then treated in the same medium without Wnt3A for 2 days. OCT4 expression was reduced and SOXl7 expression was increased in both final endoderm samples compared to undifferentiated hESCs. This differentiation analysis confirmed that hESCs cultured in suspension cultures in DC-HAIF maintained their ability to differentiate, as evidenced by similar formation of the final endoderm.

Example 15-Addition of an apoptosis inhibitor in suspension culture

Inhibitors of apoptosis were added to the medium to attenuate cell loss after initial passage in suspension. The cells were passaged as in Example 12. However, pl60-Rho-associated coiled-coil kinase (ROCK) was added to the medium instead of Y-27632.

BG02 cells in suspension population, in 3 mL DC-HAIF medium were seeded with 2 × l0 ⁶ single cells in 6-well dishes, were formed by placing 100rpm of rotating platform within the incubator (Table 2, Experiment A ). 10 μM Y27632 ROCK inhibitor was added to the test wells according to experimental design, observed daily and counted after 24 hours (1st day) and 4 or 5 days. As shown in FIG. 20, the addition of Y27632 had a tremendous effect in the early assembly phase of suspension culture. A much larger population was formed in the presence of Y27632 compared to cells assembled in inhibitor-free medium (FIG. 20). Cell counts confirmed that more viable cells were present in the presence of the inhibitor (Table 2, Experiment A). This difference in cell number persisted throughout the culture period. Also, more cells were observed on day 4 compared to cultures without inhibitors. As in the previous suspension culture experiment, cells exposed to Y27632 could be continuously passaged and maintained in an undifferentiated state (not shown). Upon re-division, approximately twice as many cells as those treated with Y27632 were observed (Experiment A in Table 2). RT-PCR analysis showed that in the presence of Y27632, BG02 cells grown in suspension culture remained undifferentiated (FIG. 21).

Experiments were performed in which Y27632 was removed after this initial period, as previous experiments showed that cell growth rates in suspension and adhesion cultures were similar after the initial 24 hours (Table). 2, Experiment B). Consistent with previous observations, Y27632 increased early survival, the assembly of hESCs after the first passage, but when the inhibitor was removed after 24 hours, the number of cells analyzed on day 5 and There was no negative effect on the viability count. Compared with 3.9 × 10 ⁶ cells in culture untreated, when the inhibitor was present, the survival of 1.4 × ^l0 7 (+ Y27632) and 1.8 × ^l0 7 (+/- Y27632) Cells have developed. This analysis confirmed that Y27632 had the greatest impact during the first 24 hours of suspension hESC culture.

Experiments were conducted to see if it was possible to reduce the number of cells used to seed suspension cultures due to the improved survival and assembly observed in the presence of Y27632 (Table 2, Table 2,). Experiment C). Previous experiments have shown that seeding of embryonic stem cells at low densities of cells below approximately 5 x 105 per 3 mL DC-HAIF does not work well. The addition of ROCK inhibitor in order to confirm whether to allow cell seeding at low density, cell concentration range (2 × 10 ⁶ cells of about 1 × 10 ⁵ cells), in 3 mL DC-HAIF 6 Well tray cells were used to seed suspension cultures. 10 microM Y27632 was added under all conditions and cell number and viability were assessed on day 5. Successful set and expansion were observed even at low sowing densities. Even medium only were seeded at 1 × 10 ⁵ cells, the expansion of approximately 13 times the viable cells was observed. Therefore, suppression of ROCK by Y27632 facilitated early survival of hESCs at lower densities in this suspension culture system.

Expt. : Experiment p0 is passage 0, p1 is passage 1 N / A, the number and proportion of unmeasured cells are rounded to the 0th or 1st place, respectively.

Example 16-Suspension culture in various media Embryonic stem cells vary with and without these factors to determine if suspension culture of embryonic stem cells is possible in the absence of FGF2 and / or Actibin A. It was cultured in a medium. Table 3 shows the cell count results for suspension cultures, which were successful in the absence of exogenous FGF2 (HAI condition) or in the absence of exogenous FGF2 or activin A (HI condition). Indicates that it was able to expand. Addition of Y27632 increased cell yield in 5 days under all conditions. In addition, the cells in each medium showed no morphological signs of differentiation and were successfully passaged.

Example 17-Optimized Shear Velocity at Increased Survival, Uniform Density, and Size of Suspended Cell Aggregates According to the systems, methods, and equipment disclosed herein, in suspended cells. All sustainable cell lines are intended to benefit and be available. Cells include, but are not limited to, human cell lines CyT49, CyT203, Cyt2S, BG0l, BG02, mice, dogs, mammal cells including non-human primate stem cell lines, and others.

The results provided herein indicate that cell proliferation and differentiation can be maintained or attenuated at controlled levels, depending on the operating parameters of the reactor, especially the flow rate and shear forces of the culture. Shown. The shear force applied to the cell culture can have a significant effect on cell proliferation. Symmetrical systems such as rotating platforms used herein provide uniform, predominantly laminar shear stresses around the vessel, and asymmetric systems such as stirrer bioreactors and their installation are local. It has a turbulent region characterized by symmetric fast shear stress. Unless the bioreactor or cell culture device is a symmetrical system, the direction of culture flow affects both the nature and degree of shear stress resulting from rotation.

Of course, the optimal number of revolutions depends on the specific medium, the number of cells in the medium, the viscosity of the culture medium, the type of medium, the toughness of certain cells in suspension (some cells are larger than others). Can withstand shearing force) and so on. The optimum number of revolutions can be easily determined according to a specific set of conditions. The rotation speeds described and assumed herein are particularly useful for maintaining laminar flow conditions. The experiments described herein were performed under the following conditions: 1) Cell proliferation and differentiation were maintained at or near controlled levels. And 2) the conditions under which cells proliferate and differentiate have been attenuated. The following is a general method capable of satisfactorily maintaining an hES cell aggregate culture or a differentiated hES cell aggregate culture. One of ordinary skill in the art can optimize the size and shape of the cell assembly based on the description provided herein.

Table 4 below shows the shear rates and stresses associated with cell assembly diameter (μm). Human embryonic stem cells were assembled for 1, 2, 3 and / or 4 days at various rotational speeds using an orbital rotator (Bamsted LabLine Multipurpose Rotator): 60 rpm, 80 rpm, 100 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, and 160 rpm. Table 4 also shows the effective shear rates experienced by the cell aggregates, which depend on the diameter of the cell aggregates.

To determine how the rotational speed controls the diameter of the ES aggregate, it was spun at 100 rpm, 120 rpm or 140 rpm to generate the ES aggregate. The diameter of the aggregate was quantified from a 5x phase contrast image obtained from the rotary culture after 2 days. In the l00 rpm culture, the average diameter +/- SD was 198 μm +/- 21 μm. In culturing at 120 rpm, the average diameter +/- SD was 225 μm +/- 28 μm. In culturing at 140 rpm, the average diameter +/- SD was 85 μm +/- 15 μm. Each diameter distribution is statistically significant using ANOVA and Tukey Multiple Comparison posttests (p <.
001). As shown in Table 4, the shear rate increases exponentially from 60 rpm to 140 rpm. For example, at 100 rpm for a 100 μm diameter aggregate, the shear rate is approximately 30 seconds -1, whereas at 140 rpm it is approximately 80 seconds -1, which is a triple increase. Typically, rotation speeds above 140 rpm resulted in higher, less uniform hES cell aggregates. The cell assembly culture is initially cultured at a reduced rotation speed, eg, 60 rpm to 80 rpm for about 1 day, and then at a higher rotation speed (eg, 100 rpm-140 rpm or higher). It can be cultured without adversely affecting its size and shape.

It should be noted that although the diameter of the cell assembly varies with shear rate, there is no significant effect on gene expression under different conditions, i.e. different rotation speeds and / or different sizes and shapes of the cell assembly. is important. That is, the signature markers observed for pluripotent hES cells or hES cell-derived cell types (eg, final endoderm, foregut endoderm, PDXl endoderm, pancreatic endoderm, and endocrine cells). It was consistent with what was described in d'Amour et al. Applications and their related applications, which are referenced and incorporated herein above.

It was shown that survival was improved by one day at a reduced rate (eg, 60 rpm to 80 rpm) to determine the effect of rotational speed, shear rate, and shear stress on cell survival or cell viability. For example, cell survival reached at least 60% or more at rotational speeds between 60 rpm and 140 rpm. Also, the number of cell aggregates was higher in the reduced rotational speed cultures d1, d2, and d3 compared to the higher rotational speeds (eg, l00 rpm and above). There was also significant division and disintegration when the cell aggregates were cultured at higher rotational speeds (eg, 140 rpm and above). However, at the same time, these data show that cell viability is increased when the cell aggregates are first cultured at a reduced rotation speed for at least one day, and when the rotation speed is increased from 100 rpm to 140 rpm, the cells It shows that there was no significant downtime in survival. However, differentiation at a rotation speed of less than 140 rpm is preferable.

Culture volume also affects shear rate and shear stress, as discussed above, which affects the size and shape uniformity of cell aggregates. For example, when single cell suspension cultures are initiated and cell aggregates are formed at 6 mL, cell aggregates of more uniform size and shape are obtained as compared to those initiated at 4 mL. See FIG. 23. When cultured in 4 mL, the diameter of the cell aggregate changes from less than 50 microns to more than 250 microns, but when cultured in 6 mL, the diameter is narrower and the diameter of the cell aggregate is from more than 50 microns to more than 200. It became less than a micron. The cell aggregates described were initiated from single cell suspension cultures made from adherent hES cell cultures, but cell aggregate suspension cultures initiated from hES-derived adhesive plating behave similarly. It is expected. Therefore, the volume of medium is largely independent of the stage at which cell assembly suspension culture is initiated.

In addition, hES cell aggregates can be cultured under a variety of different media conditions. For example, StemPro®-containing medium, DMEM / F12-containing medium; 20% Knockout serum substitute-containing (KSR, Invitrogen) DMEM / F12-containing medium; 20 ng / mL FG.
StemPro® and DMEM / F12 medium further containing F (R & D Systems) and 20 ng / mL Actibin A (R & D Systems); StemPro® and DMEM / F12 medium further containing 10 ng / mL Heregulin B. In hE
S cell aggregate culture can be maintained. Alternatively, either the medium referred to herein or a commercially available medium can be used, supplemented with a foreign-free KSR (Invitrogen). Finally, the cell aggregates were also cultivated and produced in the above mediums, which also did not contain exogenous FGF.

Example 18-hES cell aggregates in suspension culture can differentiate into endoderm linenage type cells, d'Amour et al. (2006) and US Patent Application Nos. 2005/02666554, 2005/0158553, 2006/0003313, 2006/0148081, As described in 2007/0122905 and 2007/0259421, human embryonic stem (hES) cells are maintained in vitro and the final endoderm (stage 1), anterior intestinal germ layer, and PDXl endoderm. Differentiate into. The above references are referenced and incorporated in their entirety as part of this specification.

Briefly, undifferentiated pluripotent hES adherent (plate) cells are on the mouse embryonic fibroblast feeder layer (Millipore, earlier Chemicon or Specialty Media) or in 20% KnockOut serum substitute (Invitrogen). / Gibco, 1 mM non-essential amino acids (Invitrogen / Gibco), Glutamax (Invitrogen / Gibco), penicillin / streptomycin (Invitrogen / Gibco), 0.55 mM 2-mercaptoethanol (Invitrogen / Gibco), and 4 ng / On a 60 mm plate (0.1-20% final concentration; Valley Biomedical) coated with human serum in DMEM / F12 (Mediatech) supplemented with recombined human FGF2 (R & D Systems) from mL to 20 ng / mL. Was maintained at. Alternatively, the medium can be supplemented with foreign body-free KSR (Xeno-free KSR) (Gibco) and human serum. Human serum was also added to the culture after the hES cells were seeded on an uncoated culture plate. A small dose of activin A (2-25 ng / ml, R & D Systems) was added to the growth medium to help maintain undifferentiated growth. Adhesive pluripotent hES cells on day 0 (d0) express high levels of pluripotent protein marker, OCT4. See the plate controls in FIGS. 1, panels A, d0.

The cells were manually or enzymatically repassed substantially as described in d'Amour et al., Supra. Suspended cultures were separated, transferred to a conical tube and centrifuged at 1000 rpm for about 5 minutes. Remove the supernatant and use the ViCell Analyzer (ViCell)
A standard cell count was performed using Cell Analyzer). Typical cell numbers from 60mm plates varies in the range of 3 × l0 ⁶ of 12 × 10 ^6, depending on the number of days and cell lines in culture prior to passage. Once the cell number in the primary cell suspension was measured, the suspension was further diluted with Medium Pro® or a medium containing non-foreign KSR as described above, 1 × 10 ⁶ cells / mL. Diluted to the final volume of. This volume can be increased to> 4 × 10 ⁶ cells / mL, and require more frequent feeding. The ROCK inhibitor Y27632 (Axxora) was added to the cell suspension to a final concentration of approximately 1-15 μM, typically 10 microM. The tubes were then mixed by a gentle inversion. In some cases, Y27632 was not added to the suspension to control the rate of aggregate formation. The resuspended cells were then equally distributed into each well of a low binding 6-well dish (approximately 5 mL of cell suspension per well) and at 100 rpm to 140 rpm for approximately 1-4 days prior to differentiation. Placed on a rotating platform.

During this culture, hES cell aggregates were formed into the culture by replacing 4 mL of Y27632 with 4 mL of fresh StemPro® medium minus Y27632 at least 1-2 times daily. Feeding daily or supplementing any of the above media with non-foreign KSR. Medium exchange (“feeding”) should be performed as quickly as possible because it avoids the division of the aggregates and may break the surface tension that allows the aggregates to float during rotation. Also, cell aggregates should not be removed from the rotating platform or device for extended periods of time in order to optimize cell aggregate growth and optimize aggregate size and shape uniformity. Thus, hES cell aggregates can be produced from hES cell adhesion cultures established in the art.

Currently, hES cell aggregates can be directly differentiated as aggregates in suspension, as described in D'Amour et al. (2006) above. Briefly, StemPro® (minus Y27632) medium or any of the above-mentioned medium supplemented with non-foreign KSR is removed (eg, aspirated) from the wells and 5 mL of hES cell aggregates. With 5 mL of serum-free RMPI (Cat. 15-040-CV; Mediated.), Penicillin / Streptomycin (Invitrogen), and Glutamax (also RMPI, Pen / Strep, and Glutamax medium). (Called), washed with 0% FBS, 1% pen / strep, 1% glutamax. The 6-well dish was then placed back on the rotating platform 1-2 minutes before removing the wash medium. This was repeated at least twice or until insulin and / or IGF-1 was sufficiently removed. It is necessary for maintenance of pluripotency and ES self-renewal, but because the same elements are detrimental to controlled, synchronized, linegage-derived differentiation. With the addition and removal of various exogenous mitogens, differentiation into all endoderm linenage was substantially performed at 100 rpm, as described by d'Amour et al. (2006). More details will be given below.

Differentiation into final endometrial (stage I) Human ES cell aggregates are RPMI, 100 ng / mL activin A and variable concentration FBS (US Defined FBS, HyClone, Catalog No. SH30070.03), and 25 ng / on day one. mL-75 ng / mL Wnt3a, days 2 and 3 (d0 to d2) were further differentiated in RPMI, pen / strep, and glutamax media containing 100 ng / mL activin A and variable concentrations of FBS (HyClone). It was. If a 3-day stage 1 design of experiments is used, or if desired, most differentiation experiment FBS concentrations are 0% for the first 24 hours (d1) and 0 for the second 24 hours (d2). 2% (d2), and 0.2% (d3) for the third 24 hours (d3). Desirably, a two-day stage 1 design of experiments is performed.

QPCR analysis of hES-derived cell aggregates in suspension cultures at the end of the 2nd stage 1 design of experiments is more efficient towards the final endoderm of hES aggregates compared to adherent plate controls. Shows directed differentiation. Cell aggregates were formed at 100 rpm, 120 rpm, and 140 rpm. In some experiments, hES-derived aggregates were transferred to a bioreactor (spinner flask) prior to differentiation. The final hES cell culture differentiated into endoleaf cells and the adherent hES cell culture were used as controls. Increased expression levels of SOX17 and FOXA2 were observed in suspension and adherent culture cell aggregates compared to undifferentiated hES cell aggregates and adhesive plate controls. See FIG. 22, stage 1 (d2) of panel C (SOXl7), and D (FOXA2). In addition, the expression levels of genes associated with SOX7, extraembryonic and organ endoderm contamination were significantly reduced within the final endoderm cell assembly compared to the final adherent plate control of the endoderm. See FIG. 22, stage 1 (d2) of panel L.

Flow cytometric analysis using CXCR4 and HNF3beta (FoxA2) proteins revealed that directed differentiation of ES cell-derived aggregates was at least 97% CXCR4-positive, at least 97% HNF3beta-positive, and at least 95% CXCR4 / HNF3beta. -Showed to result in a positive population.

To further assess the efficiency of hES cell assembly differentiation, frozen sections of ES-derived cell aggregates were examined for SOXl7 and HNF3beta expression using immunocytology and confocal microscopy. Image analysis of the stained frozen sections showed that nearly 90% or more of all cells at the end of stage 1 (final endochlorous cells) expressed HNF3beta and / or SOXl7.

All of these data show that highly efficient differentiation of embryonic stem cells as cell aggregates can be achieved and that they are based on the expression level of the final signature definitive endoderm marker in the discriminant region. Compared to differentiation, the method for producing the final endoderm, as described herein, shows that it is more efficient.

Differentiation into PDXI-negative foregut foliage cells (stage 2)

Aggregates of human definitive endoderm cells from stage 1 are readily washed with PBS +/+, followed by an additional 2% FBS, 25 ng-50 ng / with RMPI, pen / strep and glutamax medium. They were further differentiated for 2-3 days in those containing mL KGF (R & D Systems). In some experiments, 5 microM SB431542 (Sigma Aldrich Inc.) or 2.5 micron TGF-beta Inhibitor IV (Calbiochem) was added during the first day of stage 2. Alternatively, RMPI, pen / strep, and glutamax medium / 0.2% FBS / ITS (insulin / iron-binding globulin / selenium) were used.

QPCR analysis was performed substantially as discussed above. Increased expression levels of HNFlβ and HNF4alpha were observed in cell assembly cultures as compared to controls in adhesive plate cultures. See FIG. 22, panel E (HNFlB), and stage 2 (d5) of panel O (HNF4alpha). The method of producing specific stages 0, 1, 2, and 5 hES or hES-derived cell populations (or "dAggs" for differentiated populations) was slightly modified in Panel O. In this context, differentiated cell aggregates refer to differentiated hES or hES-derived cell aggregates initiated from the adhesive plate control culture from which they are derived at the corresponding stage. For example, in stage 1, a suspension culture of differentiated cell aggregates (“dAggs”) is initiated from the adhesive plate of stage 0 and is on a rotating platform from l00 rpm to 140 rpm for about 24 hours, the medium described herein. Incubate in any of. These differentiated cell aggregates were further differentiated into stage 1 final endoblasts on a corresponding adhesive plate control. FIG. 22, Panel O shows that there is no significant HNF4alpha (HNF4A) expression in either the aggregate of one differentiated cell at the stage or the adhesive plate control. In contrast, a similar method was performed for stage 2 samples to produce HNF4A at increased levels of expression. The expression of HNF4A is also firm in the stage 5 sample.

Moreover, the expression levels of genes associated with extraembryonic endoderm (SOX7) were significantly reduced in hES-derived cell aggregate cultures compared to plate culture controls. See FIG. 22, panel L, stage 2 (d5). The results show that directed differentiation of PDXl-negative foregut foliage cells by cell aggregates in suspension culture removes extraembryonic endoderm contaminants.

All of these data show that directed differentiation of hES cell aggregates is highly efficient, producing foregut foliage cells based on the expression level of PDXl-negative foregut endoderm markers in the discriminant region. The method for this is shown to be improved compared to differentiation in adhesive plate cultures.

Differentiation into PDXl-positive foregut foliage cells (stage 3)

Preintestinal germ layer cells from stage 2 are serum-free RMPI for 1-3 days, glutamax (Invitrogen), penicillin / streptomycin (Invitrogen), 0.5X B27-supplement (Invitrogen / Gibco), plus 1 micron or more. 2 microm retinoic acid (RA)
, Sigma) and 0.25 nM KAAD-cyclopamine (Toronto Research Chemicals); or 1 microM to 2 microM retinoic acid, 0.25 nM KA
AD-Cyclopamine and 50 ng / mL nogin (R & D systems) were added for differentiation. Alternatively, 0.2 microM to 0.5 microM RA and 0.25 nM KAAD-cyclopamine were added to the medium for 1 day. In addition, in some experiments RA or KAAD-cyclopamine was not added to the cell assembly culture. In another embodiment, 0.1-0.2% effective concentration of BSA was added.

Increased expression levels of PDXl were observed in hES-derived cell aggregates as compared to adhesive plate culture controls. See FIG. 22, panel F (PDXl), stage 3 (d8). In addition, the expression levels of genes and organ endoderm (AFP) associated with extraembryonic endoderm (SOX7) were significantly reduced in hES-derived cell aggregate cultures compared to plate culture controls. See FIG. 22, panel L (SOX7), and panel N (AFP), stage 3 (d8). Therefore, it was shown that directed differentiation to produce PDXl-positive foregut foliage cells by cell aggregates in suspension culture removes extraembryonic endoderm contaminants.

These data show that directed differentiation of hES cell aggregates is highly efficient, and PDXI-positive endoderm cells are identified based on the expression level of PDXl-positive anterior germ layer markers in the discriminant region. The method for production is shown to be improved compared to differentiation in control adhesive plate cultures.

Differentiation into pancreatic endoderm or pancreatic endocrine progenitor cells (stage 4)

In stage 4, RA was removed from the stage 3 culture and the medium was washed once with DMEM plus B27 (1: 100 Gibco). The wash solution is then replaced with DMEM + 1XB27 supplement alone or in combination with any or all of the following elements for 4-8 days: Nogin (50 ng / ml), FGF10 (50 ng / ml), KGF (25-50 ng / ml). , EGF (25-50 ng / ml), 1-5% FBS. In the absence of RA, nogin was added to the medium at 30-100 ng / mL (R & D systems) for 1-9 days. In addition, in some experiments, FGF10 was added at 25 ng / mL.

Increased expression levels of NKX6.1 and PDX-I and PTFlA were observed in adhesive plate culture controls corresponding to ES cell-derived aggregates. See FIG. 22, Panel F (PDXl), Panel G (NKX6.1), and Panel P (PTFAl), Stage 4 (d11). In FIG. 22, panel P, to determine whether hES and / or hES-derived aggregates in suspension cultures are affected by the number of cells in the adhesive bar culture from which they are derived. The bar graph represents the result from the method. Only the panel P is shows the results of 1 × 10 ⁷ cells, cell aggregates suspensions cultures different seed counts (eg, from 1 × 10 ⁶ 2 × 10 ⁷ cells) was started from. All were substantially similar, producing cell population cultures with good growth and almost no cell death. For example, in stage 4, differentiated cell aggregate suspension culture cells (“dAggs”) are started with an adhesive plate culture of d5 (stage 2) and again in any of the media described herein. Incubated on a rotating platform from 100 rpm to 140 rpm for 24 hours. These differentiated cell aggregates were further differentiated into stage 4 pancreatic endoderm-type cells expressing PTFlA (Panel P). Increased expression of PTFlA is seen as compared to the corresponding stage 4 adhesive plate culture controls.

In addition, AFP expression levels were significantly reduced in hES-derived cell aggregates compared to adhesive plate culture controls. See FIG. 22, panel N, stage 4 (d11). Therefore, directed differentiation to produce PDXl-positive pancreatic endoderm cells by cell aggregates in suspension culture removes endoderm contaminants from the organ.

Flow cytometric analysis using NKX6.1, HNF3beta and Chromogranin (CHG) proteins showed that effective differentiation of hES-derived cell aggregates was at least 53% CHG-positive and at least 40% NKX6.1 and CHG. It was shown to be co-positive and given a small amount of HNF3beta and a cell aggregate that is another type of cell.

A cold cross section of the hES-derived population was examined at the end of stage 4 using immunocytochemistry and confocal microscopy for the expression of NKX6.1, PDXl and NKX2.2. Image analysis showed highly efficient differentiation of aggregate cells into pancreatic endoderm (or PDXl-positive pancreatic endoderm). Almost all cells expressed PDXl and a large population of cells expressed NKX 6.1 (about 40% cells) and / or NKX 2.2 (about 40% cells).

Differentiation into hormone-expressing endocrine cells (stage 5)

In stage 5 differentiation, stage 4 differentiated cell aggregates were continued with either CMRL (Invitrogen / Gibco) or RMPI, pen / strep, and glutamax medium and 0.5X B27-supplement. In some experiments, the medium was supplemented with human serum (Valley Biomedical) or fetal bovine serum in the range of 0.2-5% during stage 5.

Again, increased expression of genes associated with a particular cell type was observed compared to adhesive plate culture controls, as well as cell types from stage 2-4. For example, increased expression levels of the hormone insulin (INS), glucagon (GCG), and somatostatin (SST) were observed. See Stage 5 (dl5) of FIG. 22, Panel I (INS), Panel J (GCG), and Panel K (SST). In addition, the expression levels of genes associated with AFP, ZICl, and ectoderm were significantly reduced in hES-derived cell aggregates compared to adhesive plate culture controls. See FIG. 22, stage 5 (dl5) on panel M (ZICl), and panel N (AFP). Therefore, directed differentiation to produce pancreatic endocrine cells through cell aggregates in suspension culture removes pollutants in the outer germ layer and the endoderm of the organ.

Production of hormones derived from hES-expressing endocrine population cells was confirmed by flow cytometric analysis on day 23 with the described protocol. The population is initially 5 mL
Formed in DMEM / F12 at 140 rpm and optionally contains a knockout serum substitute (KSR; Gibco / Invitrogen, compared to 0063 for consistency) or non-foreign body KSR (Invitrogen), then differentiated at l00 rpm. It was. Analysis of NKX6.1, chromogranin A, insulin, glucagon, and somatostatin protein expression revealed that the ES cell-derived population had approximately 20% NKX6.1 + / chromogranin A-pancreatic epithelium and approximately 74% chromogranin A + endocrine tissue. Indicates to include. In addition, 11% of cells express insulin, 14% express glucagon, and 11% express somatostatin. In these, 68% of insulin + cells are single positive and 70% of glucagon + cells.
Is single-positive, and 52% of somatostatin-positive cells are single-positive. The degree of positivity for single hormones exceeds the values described in adherent cultures, which were mostly polyhormonal cells.

To further assess the efficiency of aggregation differentiation into hormone-expressing endocrine cells, a cold cross section of the ES-derived population was used for glucagon, insulin and somatostatin expression during stage 5 immunocytochemistry and confocal microscopy. Was investigated using. Image analysis of 20-fold cold cross-sections showed highly efficient differentiation of aggregate cells into hormone-positive, with almost all cells expressing glucagon, somatostatin or insulin. Also, in contrast to previous adhesion culture experiments, the majority of cells in the aggregate appear to express a single hormone as it occurs in vivo during development.

Example 19-Adhesive culture from various stages can form cell aggregates and differentiate into pancreatic endoderm type cells

In the following, the production of hES-derived cell aggregates can not only be initiated from pluripotent hES cells, but the cell aggregates are initiated directly into the differentiation medium (day 0 cell aggregates) and have been differentiated or It is shown that cells can be started from cells derived from hES-, for example, cell aggregates can be produced from cells derived from stages 1, 2, 4 and 5 or hES-.

Cell assembly on day 0

Cell aggregates produced on the first day of stage 1 (d0):
Adhesive pluripotent hES cells are grown, manually or enzymatically passaged, separated, counted and pelleted, the pellet containing RMPI, pen / strep and glutamax medium, plus 100 ng / mL. activin a, 25ng / mL-75ng / mL Wnt3a, resuspended from 1x10 ⁶ cells / mL in differentiation medium containing 0.2% FBS (HyClone) to a final volume of about 1 × 10 ⁶ cells / mL Was done. This volume can be increased to> 4 × 10 ⁶ cells / mL, and require more frequent feeding. Sometimes DNase is included at a concentration of 10-50 ng / mL. The ROCK inhibitor Y27632 (Axxora) was added to the cell suspension to a final concentration of approximately 1-15 μM, typically 10 microM. In other cases, about 1: 2000 to 1: 5000 ITS (insulin / iron-binding globulin / selenium, Gibco) was added to the medium. Both Rho-kinase inhibitors and ITS were added to support cell survival. The resuspended cells were substantially distributed equally to each well of the low binding 6-well dish described above and placed on a rotating platform at 100 rpm to 140 rpm overnight. During this culture, cell aggregates of uniform size and shape were formed. As a result, the high density cultures were effectively or substantially enriched for PDXl-positive pancreatic endoderm or PDX-positive pancreatic progenitor cells. Details are provided in Example 21.

Cell aggregates produced at stage 1 d0 are then fed daily at 1-2X in differentiation medium containing RMPI, pen / strep, and glutamax medium, with an additional 100 ng / mL for the next 2-3 days. Those containing activin A and 0.2% FBS (HyClone) were fed. The subsequent steps of the protocol (stages 2-5) are substantially the same as those described above for the ES population.

Stages 1-2 and 3 days

Cell aggregates produced in stage 1 d2-d3:
Adhesive hES cells are developed and passaged substantially as described above, and then substantially as described in d'Amour et al. (2006) (above). It was differentiated into stage 1.

At the end of stage 1 (approximately d2 or d3 to differentiation protocol; final endoderm-type cells), the adherent culture was washed once with PBS +/- and pre-warmed to 37 ° C. using a 1 mL or 5 mL pipette. The cells were separated into single cells with 2 mL of Accutase for about 2-5 minutes. Next, 4 mL of 10% FBS in RMPI, pen / strep, and glutamax medium was added, and the single cell suspension was filtered through a 40 micron blue filter (BD Biosciences) and placed in a 50 mL conical tube. .. The cells were substantially counted and pelleted (centrifugated) as described above.

The cell pellet is then medium containing RMPI, pen / strep and glutamax medium, plus 2% FBS, plus DNase (50-100 microg / ml, Roche diagnostics), and 100 ng / mL activin A. Resuspended in. Alternatively, the cell pellet is resuspended in medium containing RMPI, pen / strep, glutamax, and 2% FBS, and DNase (50-100 microg / mL), 25 ng-50 ng / mL KGF (R & D Systems). It became muddy. In some experiments, 5 micron SB431542 (Sigma Aldrich Inc.) or 2.5 micron TGF-beta Inhibitor IV (Calbiochem) was included with KGF. In some experiments, Y27332 (l0 microM) was included. The resuspended cells were equally distributed to each well of the low binding 6-well dish as described above and placed overnight on a rotating platform at 100 rpm to 140 rpm to form cell aggregates of uniform size and shape. It was.

Then, the cell aggregate produced at the end of stage 1 was further differentiated. Subsequent steps of the protocol (stages 2-5) are substantially the same as the ES aggregates in Examples 17 and 18.

Stages 2, 5 to 6

Cell aggregates produced in d5-d6 in stage 2:
Adhesive hES cells are developed and passaged substantially as described above, and then substantially as described in d'Amour et al. (2006) (above). It was differentiated into stage 2. Adherent cell aggregates from stage 1 were easily washed with PBS +/+ for stage 2 with the addition of 2% FBS, glutamax, penicillin / streptomycin, and 25 ng-50 ng / mL KGF (R & D Systems). 3 in RPMI
It was day-differentiated. In some experiments, 5 micron M SB431542 (Sigma Aldrich Inc.) or 2.5 micron M TGF-beta Inhibitor IV (Calbiochem) was added during the first day of stage 2.

End-stage adherent cells of stage 2 (approximately d5 or d6 of the differentiation protocol; foregut-type cells) were separated into single cells substantially as described above, counted, and pelleted. The cell pellet then contains DMEM, pen / strep and glutamax medium, as well as IX B27-supplement and DNase (50-100 microg / ml, Roche diagnostics), FBS-free or 1-. 2% FBS, or 0.5% to 10% human serum (hS), and; 1 microM retinoic acid (RA, Sigma) and 0.25 nM KAAD-cyclopamine (Toronto Research Chemicals); or 1 microm M Or 2 microm retinoic acid, 0.25 nM KAAD-cyclopamine, and 50 ng / mL nogin (R & D systems); or 0.25 nM KAAD
-Cyclopamine and 100 ng / mL nogin; or 100 ng / mL nogin; or 0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine; or 0.2 microM to 0.5 microM RA, 0.25 nM It was resuspended in a differentiation medium containing either KAAD-cyclopamine and 50 ng / mL nogin; In some experiments, Y27332 (l0 microM) was included.

The resuspended cells were equally distributed to each well and rotated overnight at 100 rpm to 140 rpm on a rotating platform to form cell aggregates of uniform size and shape between them.

Cell aggregates produced at the end of stage 2 are further differentiated on a rotating platform, fed 1-2X daily, and for an additional 0-2 days DMEM, pen / strep and glutamax medium, and IX B27-. Supplements, 1 microM to 2 microM retinoic acid (RA, Sigma), and; 0.25 nM KAAD-cyclopamine (Toronto Research Chemicals); 1 microM to 2 microM retinoic acid, 0.25 nM KA.
AD-Cyclopamine and 50 ng / mL nogin (R & D systems); or 0.25 nM
KAAD-cyclopamine and 100 ng / mL nogin; or 100 ng / mL nogin; or 0.2 microM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine; or 0.2 μM to 0.5 microM RA, 0. Those containing either 25 nM KAAD-cyclopamine, and 50 ng / mL nogin were fed.

Then, the cell aggregate produced at the end of stage 2 was further differentiated into stages 3, 4 and 5 as explained above.

Stages 4 and 5-10 to 30 days

Cell aggregates were produced at d10-dl4 in stage 4:
Again, the adherent hES cells were substantially grown and passaged as described above and then differentiated into stage 2 as described in D'Amour et al. (2006) (above).

For stage 3, adherent cells from stage 2 were DMEM, pen / strep, glutamax medium for 1-3 days, plus IX B27-supplement, and 1 microM to 2 microm RA and 0.25 nM. It was further differentiated in medium containing KAAD-cyclopamine. In other cases, 50 ng / mL nogin was added with RA and KAAD-cyclopamine. Alternatively, 0.2 microM to 0.5 microM RA and 0.25 nM of KAAD-cyclopamine were added to the medium for just one day. In other experiments, neither RA nor KAAD-cyclopamine was added. In stage 4, cells were given 1-2X, Glutamax-supplemented DMEM, penicillin / streptomycin, and IX B27-supplement daily. As already described in Examples 17 and 18, stage 4 cells can be further differentiated into stage 5 cells.

Adhesive cultures of stage 4 (about dl0-d14 of the differentiation protocol; pancreatic epithelial and endocrine type cells) or stage 5 (about 16-30 days of the differentiation protocol; endocrine precursors and endocrine cells) are similarly. It was separated into single cells, counted and pelleted. The cell pellet was then in DMEM CMRL supplemented with Pen / Strep and Glutamax, and IX B27-supplement, DNase (50-100 microg / ml, Roche Diagnostics), and 0-2% FBS. Was resuspended in. In some experiments, Y27332 (l0 microM) was included to support cell survival. The cells were equally distributed into 6-well plates as described above and placed on a rotating platform at 100 rpm to 140 rpm for 4 hours to overnight.

In addition, as in Examples 17-19, the cell aggregates produced in stage 2 and stage 5 were enriched in pancreatic cell type as compared to the adhesive plate culture from which they were derived. For example, in one typical experiment, cell aggregates produced in stage 2 and analyzed by flow cytometry in stage 4 have at least 98% pancreatic cell type (73% Chromogranin A positive endocrine). The type consists of cells, 25% Nkx6.0l positive pancreatic endocrine (PE), and 2% non-pancreatic cells. Adhesive plate cultures from which cell aggregates are derived consist of approximately 73% pancreatic cell types (33% chromogranin A-positive endocrine cells, and 40% Nkx6.1-positive PE), and 27% non-pancreatic cell types. .. Thus, aggregates in stage 2 can enrich progenitor cells that result in pancreatic cell types and reduce non-pancreatic cell types. Similarly, in a typical experiment, cell aggregates produced in stage 5 and analyzed by flow cytometry consisted of at least 75% of chromogranin A-positive endocrine cell types, from which the cell aggregates are derived. Adhesive plate cultures consist of approximately 25% chromogranin A-positive endocrine cell types. Therefore, the aggregate at stage 5 can effectively enrich pancreatic endocrine cells.

Thus, the methods described herein not only improve the efficiency of directed differentiation of hES cells in cell assembly suspensions, but also contaminate populations (eg, ectoderm, trophozoites, within organs). Provided is a method of reducing hES-derived pancreatic cell types (or populations) having germ layers and extraembryonic endoderms and at the same time enriching pancreatic cell types (eg, pancreatic endoderms and endocrine cells).

Example 20-Effect of cell density on hES differentiation results

The following shows that changes in cell density affect differentiation outcomes in specific media and growth factor conditions. Differentiation efficiencies resulting from regulation of cell density reflect changes in the concentration of endogenously produced transfer molecules and reflect the concentration dependence of these molecules influencing cell differentiation.

Human ES cell aggregates containing d0 cell aggregates and cell aggregates derived from hES-, which were produced directly in the differentiation medium, were generated substantially as described above. Five days after differentiation through stages 1 and 2, the differentiated cell aggregates were pooled and re-aliquoted into individual wells with different seeding densities, eg, 28 mL suspension of anterior endoderm. , Stage cell aggregates were seeded or recoated at 4, 6, 8 or 10 mL / well (cell density within the 2.5-fold range). This cell distribution was performed twice and 50 ng / mL in stage 3 medium (DMEM / PenStrep / Glutamax + 1% B27 supplement (vol / vol) + 0.25 microM KAAD-cyclopamine + 3 microM TTNPB) in one set of wells. The medium containing Nogin was supplied, and the other well sets contained 25 ng / mL Nogin. Stage 3 was continued with daily medium changes for 3 days. Two cell samples were taken during the last 3 days (or about 8 days) of stage 3 for real-time QPCR analysis and again after stage 4 (or about 14 days).

The cell density and nogin concentration used during Stage 3 had different effects on the expression of pancreatic endoderm progenitor cells and / or those genes that represent endocrine progenitor cells or precursors. Briefly, there is a linear relationship between increased cell density and the corresponding increase in pancreatic progenitor cell types (eg, pancreatic endoderm, pancreatic epithelium, PDXl-positive pancreatic endoderm). For example, after stage 3 (or day 8), increased cell density corresponds to increased cell numbers of pancreatic progenitor cells, as shown by increased gene expression of PDXl and NKX6-1. See FIGS. 24A and 24B. In contrast, there was an inverse correlation between increased cell density and decreased endocrine progenitor cell type after stage 4 (or day 14). For example, as cell density decreased, decreased expression of at least NGN3 and NKX2-2 was observed after stage 3 (or day 8). See FIGS. 24C and 24D.

However, low concentrations of nogin at any density given (eg, 25 ng / mL) reduced endocrine progenitor cell types, as indicated by reduced expression of NGN3 and NKX2-2. See FIGS. 24C and 24D. The cell density-independent action of noggin in cell culture suggests that the BMP signal endogenously generated from the cell is antagonized by the exogenously added noggin. The effect of endogenously generated signals on the differentiation outcome is not limited to BMP alone, but other growth factors and / or agents secreted into the medium by cells can grow alone or extrinsically. It can have similar or contrasting effects in combination with factors and / or agents.

Example 21-Optimization of cell assembly suspension culture generated by enrichment of pancreatic endoderm or endocrine cell type

The cell composition of the hES-derived cell population is optimized for a cell type by controlling the enrichment of various growth factors and / or agents. The pancreatic cell compositions described herein are hES cell adherent cultures, d0 cell aggregates (cell aggregates were initiated from hES adherent cultures or placed directly in differentiation medium rather than pluripotent stem cell medium) , Or, as described in the previous example, were generated from single cell suspension cultures initiated from hES-derived cell assembly suspensions at various stages of differentiation. During stage 4, cell aggregates were exposed to different concentrations of NOGGIN (N), KGF (K), FGF10 (F), and EGF (E) factor groups. The cell composition of the differentiated hES cell aggregate was evaluated by flow cytometric analysis using a panel of markers containing CHGA, NKX6.1, and PDXl. Table 4 shows the total percentage of endocrine cells, pancreatic endoblasts, PDX1 + endoblasts, and non-pancreatic cells in any aggregate.

The data in Table 4 show that the resulting composition can be optimized for certain cell types by controlling the concentration and ratio of certain growth factors. For example, by lowering the concentrations of KGF and EGF (eg, 71% vs. 22.1% of K (25) E (10)), the proportion of pancreatic endoderm type cells increased compared to endocrine type cells. .. In contrast, high concentrations of KGF and EGF and inclusion of noggin and FGF10 (eg, N (50) F (50) K (50) E (50)) reduced the number of pancreatic endocrine type cells. The total number was comparable to endocrine type cells (eg 39.6% vs. 40.1%). At higher concentrations of nogin and KGF (eg, N (50) K (50)), or in the resulting aggregate, a population of endocrine type cells compared to pancreatic endoderm cell types. Increased. Also, the proportion of non-pancreatic cell types (ie, non-PDXl-positive type cells) can be reduced by reducing the amount of KGF and EGF (eg, K (25) E (10); 1.51%) or by adding growth factors. By not adding (1.53%), it can be significantly reduced.

Therefore, Table 4 shows pancreatic endoderm, endocrine, PDXl-positive endoderm or non-pancreatic cell types at least at varying concentrations of different growth factors in culture medium at a stage of differentiation (eg, stage 4). Clearly show that a population increases and / or decreases significantly.

In addition, there are other methods of enriching or purifying cell types derived from a particular hES-. This is a method for purifying endoderm AND pancreatic endoderm cells obtained from idiopathic eosinophilia syndrome cells, filed April 8, 2008, US Patent Application No. 12 / 107,020 ( METHODS FOR PURIFYING ENDODERM AND
PANCREATIC ENDODERM CELLS DERIVED FROM HES CELLS), this patent is referenced and incorporated herein. This application describes a variety of hES-cell types, including all cell types that result in stages 1, 2, 3, 4 and 5, respectively, as described in 2006 by d'Amour et al. 2005, supra. Explain how to enrich. This application uses a variety of antibodies including, but not limited to, CD30, CD49a, CD49e, CD55, CD98, CD99, CD142, CD165, CD200, CD318, CD334 and CD340.

Methods for enriching cells or cell aggregates derived from hES- are not limited to methods using antibody affinity means, allowing enrichment of certain cell types, available to those of skill in the art, or available. It can include all methods available in the future. Enrichment can be achieved by reducing one cell type or separating from another cell type or culture.

Example 22-In vivo mature pancreatic endoderm and insulin-responsive cell aggregate suspension The method of producing the cell aggregate suspension described herein is a pancreatic precursor that functions in vivo. To show that they donate cells, the above-mentioned hES-derived cell aggregates of Example 17-21 (eg, PDXl-positive endoderm, pancreatic endoderm, pancreatic epithelium, endocrine progenitor, endocrine cells, And similar) were transplanted into animals. Methods of transfer to normal and diabetic-induced animals, determination of glucose responsiveness in the animal's body, and insulin production of transplanted mature cells in vivo were substantially achieved by Kron et al. (2008). ) Nature Biotechnology 26 (4): 443-452, and US Patent Application No. 11 / 773,944, US Patent Application No. 11 / 773,944, Pancreatic Hormone Production Method (METHODS OF PRODUCING PANCREATIC HORMONES), filed July 5, 2007. It was done in a way. These are referenced and incorporated herein. Substantially the same level of human C-peptide was observed in the sera of these animals for a period similar to that found in Kroon et al., US Patent Application No. 11 / 773,944 (above).

The methods, compositions, and devices described herein represent preferred embodiments, but are exemplary and are not intended to limit the scope of the invention. Changes and other uses there may be considered by those skilled in the art, but they are within the spirit of the invention and are defined by the claims. It will be readily appreciated by those skilled in the art that various substitutions and modifications to the invention disclosed herein can be made without departing from the scope and spirit of the invention.

It is understood that in certain embodiments of the present invention, combinations of embodiments can be provided, even though they are described as different embodiments. For example, methods for making hES-derived cell aggregates in suspension culture include any endoderm linenage cell type, such as pancreatic linenage type cells, liver linenage type cells, epithelial linenage type cells, thyroid linenage cells, and thymus linenage. It is made and can be optimized to generate cells. Therefore, it is not limited to the hES-derived cell types specifically described herein. Conversely, various aspects of the invention are described as a single embodiment for brevity, but they can be done separately or can be provided as any suitable subcombination. .. For example, those skilled in the art can use hES cells and cell aggregates derived from hES- from adhesive plate cultures or suspension cultures, from undifferentiated adhesive plate cultures or suspension cultures, to differentiated adhesive plate cultures. Alternatively, the described methods for producing from suspension culture are merely exemplary and it is clear that combinations of these methods can also be used.

All publications and patents referenced herein are hereby incorporated by reference in their entirety.

The terms "essentially consisting of" as used in the claims and specification below mean to include the elements listed in connection with this phrase and are disclosed for the listed elements. Limited to other factors that interfere with or do not contribute to the activity or action. That is, the term "essentially consists of" indicates that the listed elements are required and obligatory, depending on whether or not they affect the activity and action of the listed elements. It means that other elements can or cannot exist at will.

Claims (9)

A bioreactor containing cell aggregates derived from primate pluripotent stem cells in suspension.
A bioreactor in which cell aggregates derived from primate pluripotent stem cells in suspension are generated by a method including the following steps.
(A) To provide an undifferentiated adhesive primate pluripotent stem cell culture;
(B) Dissociating the adhesive primate pluripotent stem cell culture of (a) to form a suspension of primate pluripotent stem cell single cells;
(C) Seed a primate pluripotent stem cell suspension in a defined medium that supports the undifferentiated growth of primate pluripotent stem cells, thereby primate pluripotent stem cell suspension. Forming a culture;
(D) Primitive pluripotent stem cell single cell suspension The culture is stirred with low shear stress, and the primate pluripotent stem cells substantially uniform to the primate pluripotent stem cell single cell in the suspension. Forming an aggregate;
(E) The primate pluripotent stem cell aggregate in the suspension is brought into contact with the differentiation culture conditions, and the primate pluripotent stem cell aggregate in the suspension is transformed into the primate pluripotent stem cell in the suspension. Forming cell aggregates of origin, thereby generating cell aggregates of primate pluripotent stem cells in suspension. The differentiation and culture conditions are fibroblast growth factor family, epidermal growth factor family, growth and differentiation factor (GDF) family antagonist, retinoic acid, apoptosis inhibitor, TTNPB, Rho-kinase inhibitor, TGF-β family growth factor. The bioreactor according to claim 1, comprising one or more components selected from the group consisting of inhibitors, TGF-β family growth factors, WNT factor families, and members of Notch processing or cleavage inhibitors. The primate pluripotent stem cell aggregate
a) Differentiated or differentiated in an environment without feeders, matrices, and adhesive growth cultures
b) Rotated at about 60 rpm to 160 rpm,
The bioreactor according to claim 1 or 2. Claims 1 to that the stirring of the primate pluripotent stem cell single cell suspension culture comprises rotary culture.
The bioreactor according to any one of 3. The bioreactor according to any one of claims 1 to 4, wherein the adhesive primate pluripotent stem cell is a human embryonic stem cell. The bioreactor
a) Scale-up system or
b) Containing or containing cell aggregates derived from primate pluripotent stems that are substantially uniform in size, shape, or both.
c) The bioreactor according to any one of claims 1 to 5, which comprises a container containing a fluid nutrient medium in which a cell aggregate derived from the primate pluripotent stem is suspended. The bioreactor according to any one of claims 1 to 6, wherein the cell aggregate derived from the primate pluripotent stem is suspended in a physiologically acceptable medium. The bioreactor according to claim 7, wherein the medium does not substantially contain insulin, IGF-1, or both. The cell aggregate derived from the primate pluripotent stem
a) Express one or more markers selected from the group consisting of Brachyri, SOX17, FOXA2, HNF1β, PDX1, NKX6.1, NKX2.2, INS, GCG, SST, SOX7, and PTF1A, or
b) A cell selected from the group consisting of endoderm, ectoderm, and mesoderm, or
c) A cell aggregate selected from the group consisting of an endoderm endoderm cell aggregate, a progenitor endoderm cell aggregate, and a PDX1-positive endoderm cell aggregate, or
d) Batch feed,
The bioreactor according to any one of claims 1 to 8.