JP2016163584A - Stem cell aggregate suspension composition and method of differentiation thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium that can culture human embryonic stem (hES) cells while maintaining their pluripotency; and a human embryonic stem cell aggregate cultured in the culture medium.SOLUTION: A culture medium contains heregulin being an ErbB3 ligand and a basal nutrient salt solution, and does not contain serum. Also provided is a human embryonic stem cell aggregate cultured in the culture medium with the pluripotency maintained.SELECTED DRAWING: Figure 23

Description

Federally sponsored research or development statement Part of the work to complete the present invention was funded by the US National Institutes of Health Grant No. Made using 5R24 RR021313-05. Accordingly, the US government has certain rights to the invention.

BACKGROUND OF THE INVENTION
The present invention relates to a suspension cell assembly composition that is substantially free of serum and feeders, and a method for differentiating a cell assembly suspension.
To date, there has been no efficient system to provide a large-scale manufacturing process (“scale-up”) for eukaryotic mammalian cells, particularly mammalian pluripotency such as human embryonic stem (hES) cells. There was no cell at all. In order to maintain hES cells in an undifferentiated state in vitro, hES cells are typically maintained on a mouse fetal fibroblast (MEF) feeder and subjected to manual mechanical dissociation (eg, microsurgery). It was then passaged to individual colonies. These methods are sufficient for research studies that do not require mass production of undifferentiated or differentiated hES cells, gene targeting, drug delivery, and in vitro toxicity studies. Future clinical applications for stable and large-scale expansion of hES cells including enzyme passages require improved methods.

  Although enzyme expansion of hES cells is feasible, these methods are technically flawed because hES cells depend on cell-cell interactions as well as para- and autocrine signals for survival. Therefore, hES cells prefer an intracellular microenvironment compared to the case where they exist as single cells. There are also reports that enzyme dissociation of hES cells can lead to abnormal karyotypes, resulting in genetic and epigenetic changes. The result is a highly supportive culture environment that ensures the robustness of undifferentiated or differentiated hES cells without compromising on long culturing periods, pluripotency, pluripotency or genetic stability. It is essential to take into account the large scale expansions (ie, manufacturing processes).

Human pluripotent cells investigate the early stages of human development and provide a rare opportunity for therapeutic involvement in several medical conditions such as diabetes and Parkinson's disease. For example, the use of beta cells to produce insulin derived from human embryonic stem cells (hESCs) would provide a significant improvement over current cell therapy procedures that utilize cells from donor pancreas. Currently, cell therapy for diabetes utilizing cells from donor pancreas is limited by the lack of high-quality islet cells needed for transplantation. Cell therapy for type I diabetic patients requires transplantation of approximately 8 × 10 ⁸ pancreatic islet cells (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)
Therefore, at least two healthy donor organs are necessary to obtain sufficient islet cells for successful organ transplantation.

  As a result, embryonic stem (ES) cells represent a powerful model system for studying the mechanisms underlying pluripotent cell biology and differentiation in early embryos. It also provides the opportunity for genetic manipulation of mammals and the resulting commercial, medical and agricultural applications. Furthermore, the potential proliferation and differentiation of embryonic stem cells is used to create an unlimited source of cells that are compatible for transplantation for the treatment of diseases resulting from cell damage or dysfunction. Early primitive ectoderm (EPL) cells described in International Patent Application WO 99/53021, derived in vitro or ex vivo ICM / upper blastoderm layer, derived in vitro or ex vivo primitive ectoderm, primordial germ cells ( Other pluripotent cells and cell lines, including EG cells), teratocarcinoma cells (EC cells), and pluripotent cells induced by dedifferentiation or nuclear transfer, have some of these properties and uses Or will share everything. International patent application WO 97/32033 and US Pat. No. 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. Human EG cells are described in International Patent Application WO 98/43679.

  The biochemical mechanisms that regulate embryonic stem cell pluripotency and differentiation are very poorly understood. However, the limited experimental data available (many case evidence) show that the continuous maintenance of pluripotent embryonic stem cells under in vitro culture conditions is dependent on cytokin and growth factors present in the extracellular environment. Suggests that it depends on the existence of

Conditions that are equivalent to expected regulatory guidelines that determine clinical safety and efficacy, while human ESCs provide a source of starting material to develop significant quantities of high quality differentiated cells for human cell therapy These cells must then be obtained and / or cultured.
Such guidelines will require the use of chemically defined media.
Development of such chemically defined / GMP standards is necessary to facilitate the use of cells obtained from hESCs and hESCs in the human body for therapeutic purposes.

  Moreover, the ultimate use of hESC-based cell replacement therapy will require the development of differentiation conditions corresponding to methods and regulatory guidelines that allow large-scale culture. Several groups have reported simplified growth conditions for hESCs, but these studies have considerable limitations. In general, however, successful isolation of pluripotent cells, long-term clonal maintenance, genetic manipulation, and germ line transmission have been difficult.

  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; Beattie 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 very purified source but contains a crude product of bovine serum albumin (BSA). Others only perform short-term tests, so it is not clear whether these conditions allow maintenance of pluripotency for long periods of time. (Sato et al. (2004) Nature Med., 10: 55-63; US Patent Publications 2006/0030042 and 2005/0233446). Others have shown long-term maintenance of pluripotency in chemically defined media with FGF2, activin A, and insulin, but the cells are plates coated with human serum and plated with cells. Raised on what 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 whether it is absolutely necessary; especially some formulations In the product, it is necessary to use it at 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 a level of microg / mL, or contain insulin present by use of KSR.
It is usually believed that insulin functions in glucose metabolism and “cell survival” signaling through binding to the insulin receptor.
At levels above physiological concentrations, however, insulin can bind to the IGF1 receptor with low efficiency and give classical growth factor activity through the PI3 kinase / AKT pathway. The presence / requirement of insulin at such high levels (micro g / mL level) in KSR or other media manifests a major activity through binding to the IGF1 receptor expressed by hESCs (Sperger et al., 2003 PNAS, 100 (23): 13350-13355). Expression of intracellular signaling pathway members in the complete complement of IGFlR and hESCs is likely to imply a functional activity of this pathway (Miura et al., 2004 Aging Cell, 3: 333-343). The major signals that insulin or IGFl are required for self-renewal of hESCs can be revealed. This is suggested by the fact that all conditions developed for the culture of hESCs include insulin, insulin provided by KSR, or IGF1 provided by serum. In support of this concept, it has been shown that cells are differentiated when PI3 kinase is inhibited in hESC culture (D'Amour et al., 2005 Nat. Biotechnol., 23 (12): 1534-41; McLean et al., 2007 Stem Cells 25: 29-38).

  A recent publication reviews humanized, defined media for hESCs (Ludwig et al., Nature Biotechnology, published online Journal 1,2006, doi: 10.1038 / nbtl 177). However, this recent formulation contains several elements that are 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), neuregulin-β (also heregulin-β (HRG-β), glial growth factor, etc. HRG-α, amphiregulin, betacellulin, and epiregulin, but are not limited thereto. All of these growth factors are expressed as transmembrane proteins that generate the soluble ectodomain growth factor that contains the EGF domain and is first processed by a metalloproteinase (specifically Adam: ADAM) protein. Alternatively, members of the EGF family interact with homo- or hetero-dimers of ErbB1, 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 high affinity (range 1-100 nM) to ErbB1 / 1 (EGFR) homodimers, whereas HRG-β has very high affinity (range <1 nM) with ErbB3 and ErbB4. Join with. Active ErbB receptors signal the PI3 kinase / AKT and MAPK pathways. ErbB2 and ErbB3 are the most expressed growth factor receptors in hESCs (Sperger et al., 2003 PNAS, 100 (23): 13350-13355). 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). Furthermore, overexpression of ErbB2 and subsequent inappropriate activation is associated with the tumor development process (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 that have been observed to accumulate as trisomic of several hESCs (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; 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. , Natu re Biotechnology, published online January 1, 2006, doi: 10.1038 / nbtl 177).

  ErbB2 and ErbB3 were expressed in mouse blastocysts (Brown et al., 2004 Biol. Reprod., 71: 2003-2011; Salas-Vidal & Lomeli, 2004, Dev Biol., 265: 75-89). However, clearly not restricted to the inner cell mass (ICM), ErbB1, 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 culture (Martin et al., 1998 Hum. Reprod., 13 (6): 1645-52; Sargent et al., 1998 Hum. Reprod. 13 Suppl 4: 239-48). . And has an additional effect on mouse embryonic stem cells in 15% serum (Heo et al., 2005. Am. J. Phys. Cell Physiol., In press). The phenomenon before or immediately after transplantation includes ErbB2 − / −, ErbB3 − / −, Neuregulin 1 − / − (Britsch et al., 1998 Genes Dev., 12: 1825-36), ADAM17 / − (Peschon, Et al., 1998 Science, 282: 1281-1284), and ADAM 19 − / −, (Horiuchi, 2005 Dev. Biol., 283 (2): 459-71) do not appear to be affected in null embryos. Thus, the importance of signaling through the ErbB receptor family of hESCs has so far been unclear.

  Neuregulin-1 (NRG1) is a large gene that exhibits multiple splicing and protein processing variants. This generates many protein isoforms. (Protein isoforms are collectively referred to herein as neuregulin). Neuregulin is predominantly expressed as a cell surface transmembrane protein. 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). Cell membrane metalloproteases ADAM17 and ADAM19 have been shown to process the transmembrane form of neuregulin-1 to soluble neuregulin / heregulin. HRG-α and -β are cleaved ectodomains of neuregulin including EGF and other domains. Since the EGF domain is responsible for ErbB receptor binding and activation, 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). In addition, through the interaction between the ErbB receptor and the EGF domain, there is a processed transmembrane isoform of neuregulin that appears to trigger juxtacrine signaling in adjacent cells.

International patent application WO99 / 53021 International patent application WO 97/32033 US Patent No. 5,453,357 International patent application WO00 / 27995 US Patent No. 6,200,806 International patent application WO 98/43679 US Patent Publication 2006/0030042 US Patent Publication 2005/0233446

Shapiro et al., 2000, N Engl J Med 343: 230-238 Shapiro et al., 2001a, Best Pract Res 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. Beattie 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 Med. , 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, published 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. et al. 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, published 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. et al. Phy. Cell Physiol. , In press Britsch et al., 1998 Genes Dev. , 12: 1825-36 Peschon, et al., 1998 Science, 282: 1281-1284. Moriuchi, 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 progression of hESC research to maintain pluripotency in culture is the elucidation of media and cell culture conditions that are compatible with expected regulatory guidelines that determine clinical safety and efficacy. Will be. The best result will be the utility of chemically defined media for hESCs, but components that are not chemically defined are acceptable if they are manufactured to GMP standards. Accordingly, a method and composition 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, wherein the composition is essentially free of serum.

The invention also relates to a composition comprising a basic nutrient solution and means for stimulating ErbB2-derived tyrosine kinase activity in differentiable cells.
The present invention relates to a method for culturing differentiable cells. This method plates differentiable cells on the cell culture surface and provides a basal nutrient solution for the differentiable cells to provide a ligand that specifically binds ErbB3.

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

  The present invention also relates to a method for culturing differentiable cells, wherein the digestion solution is provided to the differentiable cell layer contained in the medium chamber before digestion, and the digestion cell layer is a single cell. Relates to a method comprising: After digestion, single cells are placed in a new tissue culture chamber with a differentiable cell culture. There, the differentiable cell culture medium contains a basic nutrient solution and an ErbB3 ligand. Once cultured, single differentiable cells are subjected to conditions that allow single cell growth and division.

The present invention relates to a method for generating hES cell aggregates in suspension from pluripotent hES adhesion media. In this method, hES cells are cultured under adherent growth culture conditions that allow expansion in an undifferentiated state;
Separating adherent hES culture cells into a single cell suspension culture;
A single cell suspension culture is agitated until the single cell suspension culture forms an hES-derived cell aggregate in the suspension, thereby producing an hES-derived cell aggregate (hES- in suspension). derived cell aggregates) in contact with a first differentiable culture condition that allows to form,
Generate hES-derived cell aggregates in suspension.
In a preferred embodiment, stirring of 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 hES-derived single cell suspension. This method comprises culturing hES cells under adherent growth culture conditions that allow expansion in an undifferentiated state;
contacting undifferentiated hES cells with a first differentiation culture condition suitable for differentiating hES cells to obtain adherent hES-derived cells;
Separating adherent hES-derived cells into single cell suspension cultures;
The single cell suspension culture is agitated until the single cell suspension culture forms hES-derived cell aggregates in the suspension, thereby allowing the single cell suspension culture to Contacting with a second differentiation culture condition allowing the formation of hES-derived cell aggregates, generating hES-derived cell aggregates in suspension. In a preferred embodiment, the stirring of the single cell suspension culture is performed at about 80 rpm to 160 rpm.

  The present invention also optimizes the cell concentration of pluripotent cell cultures, or various growth factors such as FGF10, EGF, KGF, noggin and retinoic acid, apoptosis inhibitors, Rho-kinase inhibitors, And to altering the concentration of the resulting cell culture and / or altering the population of hES-derived cell aggregate suspensions by varying the concentration of and the like.

FIG. 1 shows real-time RT PCR of ADAM19, neuregulin 1 and ErbB1-3 in BG01v in defined conditions (8 ng / mL FGF2, 100 ng / mL LR-IGF1, 1 ng / mL Activin A). The expression analysis shows GAPDH and OCT4 control reaction.

FIG. 2 shows growth inhibition of BG01v cells using AG879. BG01v cells were plated in 6-well trays and, after plating, exposed to DMSO (A), 50 nM-20 microM AGl478 (B), or 100 mM-20 microM AG879 (C) for 24 hours. It was. After 5 days in culture, the culture was 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 Activin A, 200 ng / mL LR-IGF1, and 8 ng / mL FGF2. A and B) will be described. Also shown is the morphology (C and D) of BG01v cells cultured in defined media 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 1 and ErbB1-4 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 × 10 ⁵ Mouse RlES cells were plated on 1: 1000 MATRIGEL®, 10% KSR and 1000 U / mL mouse LIF (ESGRO) in 10% FBS. The next day, DMSO (control carrier), 1-50 microM AG1478, or 1-50 microM AG879 was added along with fresh media. The medium was fixed on day 8 and stained for alkaline phosphatase activity. DMSO (A) and 1-50 microM AGl478 (B and C) did not clearly inhibit proliferation. AG879 may substantially inhibit cell growth at 50 microM (comparison of D and F) and may slow growth at 20 microM (E).

FIG. 6 shows the growth inhibition of BG02 cells grown in conditioned medium (CM). (A) 50 microM AG825 inhibited the growth of BG02 hESCs grown in CM. (B) AG825 suppresses ErbB2 Y1248 phosphorylation in hESCs. (C) Colony count of serial passages of CyT49 hESCs in different combinations of growth factors. (D) Cell count analysis of the role of IGFl and HRG in hESC proliferation using BG02 cells (left). (E) Repeated OCT4 / DAPI immunostaining showed that IGFl and HRG significantly increased the proportion of OCT4 ⁺ cells compared to ActA / FGF2 conditions. (F) RTK blotting analysis of BG01 DC-HAIF hESCs pulsed with DC-HAIF for 15 minutes overnight in starvation without growth factors. Alternatively, a steady state culture is shown (left). Normalized relative intensity means and ranges were plotted (right).

FIG. 7 shows mouse ES cells grown under defined conditions according to different growth factor combinations. (A) Shows AP ⁺ colony scores after 2 × 10 ⁵ cells have been grown for 8 days with different growth factor combinations. (B-G) shows 4 × images of AP ⁺ colonies grown with different growth factor combinations.

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 multipotent differentiation potential into ectoderm, mesoderm and endoderm. (B) Immunostaining of BG02 cells cultured in differentiated 15% FCS / 5% KSR. (C) High Density Illumina Centrix Human- containing 47,296 transcription probes in BG02 cells maintained in CM (passage 64) or DC-HAIF (passage 10 or 32 in defined medium) 6 Venn diagram of distribution of transcripts investigated using Illumina Sentrix Human-6 Expression Beadchips. (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 final passages with DC-HAIF Scatter plot analysis showing that it was not (bottom). (E) Hierarchical strain formation dendrogram of relative gene expression in different populations created using Beadstudio 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 single cell passage of human embryonic stem cells. (AD) Image of BG02 cells per stage after passaging with ACCUTASE® and plating of approximately 5 × 10 ⁵ cells in 60 mm culture ridges. (A) Shows viable cells adhere to the dish after 1.5 hours of initial plating. (B) At 20 hours after plating, the majority of cells assembled and formed small colonies. These colonies grow and expand up to 4 days after plating (C), and form a monolayer like epithelium while covering the entire dish after 5-6 days (D). (E) Normal male karyotype shown in BG02 culture passaged 19 times with ACCUTASE® in DC-HAIF.

FIG. 11 shows single human embryonic stem cells using (A) ACCUTASE®, (B) 0.25% Trypsin / EDTA, (C) TrypLE, or (D) Bercene. The cell morphology after cell passage is shown.

FIG. 12 shows large scale growth of human embryonic stem cells cultured in DC-HAIF. (A) Flow cytometric analysis of BG02 cells after expansion to> 10 ¹⁰ cells. > 85% of cells expressed OCT4, CD9, SSEA-4, TRA-1-81. (B) RT PCR analysis of expression of pluripotency markers of OCT4, NANOG, REX1, SOX2, UTF1, CRIPTO, FOXD3, TERT, and DPPA5. Markers of differentiated lineages, α-fetoprotein (AFP), MSXl, and HANDl were not detected. (C) Fluorescence in situ hybridization (FISH) using human chromosome-specific repeats showed maintenance of standard copy numbers of hChr12, 17, X and Y.

FIG. 13 shows the morphology (A) and normal karyotype (B) of hESC BG02 cells grown in defined medium containing HRG-β and IGF1 in the absence of FGF2 for periods longer than 7 or 2 months. ).

FIG. 14 shows a scatter plot analysis of transcription from hESCs (BG02) maintained in DC-HAIF (32s) or DC-HAI (10s). Most transcript expression was detected in both samples. Transcriptivity was not substantially changed by culturing hESCs in the absence of exogenous FGF2. The correlation coefficient (R2) was determined by using all detected transcripts with expression levels> 0 (all dots) or with transcripts showing a detection confidence of> 0.99 (R2). select, indicated by a dotted oval). The diagonal lines indicate the limits of the mean and 2-fold difference.

FIG. 15 shows a hierarchical strain formation dendrogram of relative gene expression in different populations of early and late passaged BG02 cells maintained with DC-HAIF. Cells clustered closely (about 0.0075), and BG02 and BG03 cells were similarly maintained (about 0.037) in conditioned medium (CM). Also, BG02 cells maintained in DC-HAI are clustered closely with other evaluated hESC populations. For illustration, in FIG. 15, CM is a conditioned medium. DC is a 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. Phase contrast imaging (A) and (B) alkaline phosphatase staining of BG02 cells (10 ⁴ cells / well) grown in one well of a 96-well plate. Phase contrast imaging (C) and (D) alkaline phosphatase staining of BG02 cells (10 ³ cells / well) grown in one well of a 384-well plate.

FIG. 17 shows a dark field image of BG02 grown in suspension culture with DC-HAIF. Shown on day 2 and day 6 cultures. Images were obtained using 4x magnification.

FIG. 18 shows DC-HAIF adhesion and growth rate in suspension culture. 1 × 10 ⁶ cells of BG02 were plated in parallel wells in adherent and suspension cultures. Cell counts were counted in 1-6 days.

FIG. 19 represents qPCR analysis of suspended and adherent hESCs. BG02 cells growing in suspension (S. hESCs) and those growing in adhesion medium (hESCs) showed comparable levels of OCT4 expression and lack of SOX17 expression. Adherent cells that have differentiated into the final endoderm (DE), and suspended hESCs that have differentiated into the final endoderm (S.DE d3) in suspension, together with the expected low expression of OCT4. A high expression of SOX17 was shown.

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 first and third days.

FIG. 21 shows RT PCR analysis of suspension aggregates in the presence of Y27632. RT-PCR was performed on the grown cultures to assess the expression of pluripotency markers. OCT4, NANOG, REX1, SOX2, UTF1, CRIPTO, FOXD3, and TERT AND DPPA5 were detected. Markers of differentiated lineage, AFP, MSX1, and HAND1, were not detected.

22A-N shows 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 pattern of PTFlA (panel P). This is not a complete list, but pluripotent human embryonic stem (hES) cells (stage 0, d0), final endodermal cells (stage 1; d2), PDX1-negative foregut endoderm Cells (stage 2; d5), PDX1-positive endoderm cells (stage 3, d8), pancreatic endoderm cells (stage 4; d11), pancreatic endocrine precursors, and / or hormone secreting cells (stage 5; d15) ) Can be used to identify.

FIG. 23 is a graph showing the range of cell aggregate diameters (microns) in suspension correlated with the total volume (mL) of medium in the culture.

FIGS. 24A-D show the hES cell culture from which PDXl (panel A), NKX6.1 (panel B), NGN3 (panel C), and NKX2.2 (panel D) in hES-derived cells are derived. It is a bar graph which shows the expression pattern of a marker gene in correlation with a cell concentration.

DETAILED DESCRIPTION OF THE INVENTION The methods described herein are in contrast to previously known methods of regenerative medicine that are based on seeding individual cells within a polymer scaffold, matrix, and / or gel. Uses a pluripotent hES single cell suspension or a cell aggregate suspension formed from an hES-derived (differentiated) single cell suspension as a building block for tissue formation. Cell aggregates are often composed of hundreds to thousands of individual cells, connected via adhesive junctions and extracellular matrix, and contribute to the final differentiated product as a whole. In this regard, a cell aggregate can be defined as a tissue type that provides many useful properties for more traditionally designed tissues.

  In one embodiment of the invention, a method is provided for 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 are initially cultured on a fibroblast feeder or they can be cultured without a feeder. The method for isolating hESC and culturing on human feeder cells is a method of culturing human ES cells on human feeder cells (METHODS FOR THE CULTURE OF HUMAN EMBRYONIC STEM CELLS ON HUMAN FEEDER CELLS). 432, 140, the entirety of which is hereby incorporated by reference. Multipotent ES cell aggregate suspension cultures made directly from or initiated from hESCs cultured on feeders avoid the need for hESC monolayer creation, for example, in adherent cultures. These methods are described in detail in Examples 17 and 18.

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

  Yet another embodiment of the present invention is a differentiated hES cell culture (hES-derived), eg, cells from stages 1, 2, 3, 4 as described in d'Amour 2005 and 2006 above. A method of producing a cell assembly suspension formed from a “cell culture” or “hES-derived cell”) is provided. 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 methods of use 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 endoderm cells, pancreatic endocrine cells, and / or PDX1 endoderm cells, different concentrations of growth factors. Do by changing. These methods are described in detail in Example 21.

  Unless otherwise stated, the terms used herein should be understood according to the conventional usage of those skilled in the relevant 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 Glossary of genetics: classical and molecular, 5th Ed. , Berlin: Springer-Verlag; and Current Protocols in Molecular Biology, F .; M.M. Ausubel et al., Eds. , Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. , (1998 Supplement). Where the singular form is used in the specification and claims, it should be understood that it has one or more meanings based on the context in which it is used. For example, “cell” means at least one cell.

  In the specification and claims, unless otherwise specified, the amounts of ingredients, ratios or ratios of substances, reaction conditions and other numerical values are understood to be modified by the term “about” in all cases. Is done. Unless stated to the contrary, the numerical parameters set forth in the specification and claims are approximate and may vary depending upon what is required to be obtained by the present invention. At least, it does not limit the application of the doctrine of claims, and each numerical parameter is reported as at least a significant figure and is based 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 factors have been standardized that can be used for cell movement and assembly in culture, hydromechanical microenvironments that correlate with optimal cell aggregate viability and properties, and scale-up Because it affects variables, it is necessary to characterize the growth and differentiation behavior and effects of cells in various culture vessels, dishes, bioreactors, etc., and the 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 rate and shear stress are mechanical properties that define fluid shear in the system. Shear rate is defined as the fluid velocity at a particular distance and is expressed in units of seconds ⁻¹ . The shear rate is proportional to the shear stress, and shear rate (γ) = shear stress (t) / viscosity (micro). Shear stress is defined as the fluid shear force acting in the tangential direction of the cell surface and is expressed as force per unit area (dyne / cm ² or N / m ² ). Shear stress is generated by agitating a fluid containing static cells, by agitating cells through a static fluid, or by moving cells through a dynamic fluid environment that has been agitated be able to. Fluid viscosity is typically measured in poise, where 1 poise = 1 dyne seconds / cm ² = 100 centipoise (cp). One of the least known viscous fluids, the viscosity of water is 0.01 cp. The viscosity of a typical suspension of eukaryotic cells in the 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 and uniform streamlines at low speed. In contrast, turbulence is dominated by high velocities and inertial forces, characterized by the occurrence of eddies and vortices, and chaotic flow across space and time. A dimensionless number known as the Reynolds number (Re) is commonly used to quantify the presence of laminar or turbulent flow. The Reynolds number is the ratio of inertia to viscous force and is quantified as (density × speed × length scale) / (viscosity). The laminar flow is dominant when Re <2300, and the turbulent flow is dominant when Re> 4000. Based on this correlation with fluid velocity, the Reynolds number and its consequences, ie, whether the fluid flow is laminar or turbulent, depends on the shear rate and shear stress experienced by the cells in suspension. Directly proportional. However, high shear stress conditions can occur in both laminar and turbulent liquid environments. Initially, the liquid tends to resist movement. For fluids closest to a solid surface subject to attraction, a boundary layer or no flow area occurs immediately adjacent to the surface. This creates a fluid velocity gradient from the surface to the center of the fluid flow. The slope of the fluid velocity is a function of the distance from the boundary layer to the region of highest fluid velocity and the 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, laminar slope is broken and turbulence is formed. Thomas et al. Have shown that cell degradation under turbulent conditions occurs most frequently in the region of locally high shear stress and high energy dissipation rate. 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 very high shear stress regions that can have a detrimental negative impact on the scale-up of cell culture manufacturing systems. Therefore, there is a need for a method that can maintain cell density and viability in mammalian cell culture production scale-up systems by controlling the main source of shear force of such systems.

Henzler (Henzler, 2000, Particle stress in bioreactors), In Advances in Biochemical Engineering / Biotechnology, Scheper, T. Ed. Springer-Verlag, Co. et al. Bulk fluid in rotating 6-well dishes according to the method given by Experimental analysis of coagulation of particles under low-shear flow, Water Res. 39: 2994). The fluid mechanical properties of were calculated. The dimensionless stress is equal to the turbulent constant × (aggregate diameter / Kolmogorov microscale) ^Λ turbulence index. Shear stress is equal to dimensionless stress x fluid density x (kinematic viscosity x force input) ^0.5 . The shear rate is equal to the shear stress / kinematic viscosity. In force input and Kolmogorov microscale calculations, the Reynolds number is required for each rotational speed and is equal to (rotational speed x flask diameter) ^Λ 2 / viscosity. Since both the force input and the Kolmogorov microscale are functions of the Reynolds number, all shear stresses and shear rate calculations vary according to the rotational speed.

Moreover, shear stress and shear rate are a function of dimensionless stress that depends on the diameter of the formed aggregate, so that the shear stress and rate experienced by the aggregate are expected to increase with the time of rotation. . An example calculation is shown in Example 17 for an assembly diameter of 100-200 microns and a rotational speed between 60-140 rpm. These methods were used to provide an estimate for the average shear of the bulk fluid over time. However, the shear stress at the container wall is expected to be highest due to boundary effects. To predict wall shear stress, Ley et al. Suggested that the wall shear stress of a 6-well dish is equal to the radius of rotation x (density x kinematic viscosity) (2 x pi x rotation speed) ³ ) ^0.5 Did. Using this approach, wall shear stress was calculated for rotational speeds from 60 rpm to 140 rpm and is shown in Example 18. It should be noted that unlike the time averaged shear stress experienced by an aggregate in a bulk fluid, the shear stress occurring at the wall is 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 traditional tissues that are two-dimensional (eg, constructed from glued together). The effect on cell concentration differentiation is explained in more detail in Example 20. By assuming a three-dimensional (3D) structure that more accurately reflects cell density and conformation in vivo, cell aggregates can overcome this limitation. As a result, the period of time for cells to achieve their intended structure can be significantly reduced and / or made more consistent and efficient. Moreover, 3D assembled cells can differentiate and function more optimally. This is because this structure resembles a more normal physiological phenomenon than adhesion culture. Mechanical difficulties in the manufacturing process cause little damage to floating cell aggregates in suspension culture, for example compared to mechanical difficulties in adhesion culture.

  Also, typical production scale suspension cultures utilize continuous perfusion of the medium as a method to maintain cell viability while maximizing cell concentration. In such a relationship, medium exchange provides different effects of fluid shear on adherent cells and suspension aggregates. When the media stream flows across the cell surface, the immobilized adherent cells may undergo fluid shear stress. In contrast, suspension assemblies are subject to little shear stress across the assembly surface. This is because the aggregate can freely collapse in accordance with the applied shear force. It is expected that long term shear stress is detrimental to adherent embryonic stem cells and that the suspended aggregate format 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 above related to shear rate and shear stress There is a need based on the observed need for mechanisms. The present invention provides a method for producing pluripotent stem cells and / or pluripotent progenitor cells obtained for the first time from pluripotent stem cells in a suspension format, particularly a cell aggregate suspension format. I will provide a.

  As used herein, “single cell suspension” or equivalent expression thereof, in all mechanical or chemical meanings, is hES cell single cell suspension or hES-derived (hES -derived) means single cell suspension. There are several ways to dissociate cell clumps and form single cell suspensions from primary tissues, adherent cells and aggregates in culture, such as physical forces (cells Mechanical dissociation, such as scrapers, narrow-bore pipettes, fine needle aspiration, vortex shedding, and forced filtration through fine nylon or stainless meshes, enzymes (trypsin, collagenase, acutase, etc.) Dissociation), or a combination of both. In addition, methods and culture medium conditions that help to support the dissociation of hES cells into single cells are useful for defining seeding for expansion, cell sorting, multiwell plate assays, culture procedures and clones. Enables automation of expansion. Thus, one embodiment of the present invention is a stable enzymatic dissociation hES of single cells that can support long-term maintenance and efficient expansion of undifferentiated multipotent or differentiated hES cells. Methods are provided for generating cells or hES-derived cell culture systems.

As used herein, the term “contact” (ie, the contact of a cell, eg, a differentiable cell, with a compound) refers to the compound and cell together in vitro (eg, the compound in culture). Is added to the cells).
The term “contact” refers to the in vivo exposure of a cell to a defined cellular medium that includes an ErbB3 ligand, and optionally a member of the TGF-β family, that may occur naturally in a subject (ie, natural It is intended not to include exposures that may occur as a result of the physiological process. The step of contacting the cell with a defined cell culture medium comprising an ErbB3 ligand and optionally a member of the TGF-β family can be performed in any suitable manner. For example, the cells can be treated in adherent culture or suspension culture. It will be appreciated that cells that have been contacted with a defined medium to stabilize or differentiate 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 encompasses partially or ultimately differentiated cell forms. In general, 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 large range or degree. However, this method is different from other methods. As used herein, for example, “substantially free”, eg, “substantially free of contaminants”, “substantially free of serum”, “substantially insulin or insulin-like growth factor” Not contained in, or equivalent terms thereof, is at least 98%, at least 98.5%, at least 99%, or at least 99.5%, or at least 100% of solutions, media, supplements and excipients. Means free of 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 is described as “substantially similar” or equivalent expression, 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 the methods incorporated herein in its entirety. Similar to that.

  In certain embodiments of the invention, the term “enriched” means that the desired cell lineage is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% Or 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 in the absence of serum or serum replacement. Refers to a sufficient concentration of a compound present in the remaining components of a defined medium to affect cell stabilization. This amount can be readily determined by one skilled in the art.

  As used herein, the term “expression” refers to the transcription of a polynucleotide or the translation of a polypeptide in a cell, and the level of the molecule is measured in cells that express the molecule compared to cells that do not express the molecule. To be made as high as possible. Methods for measuring molecular expression are well known to those of skill in the art and include, but are not limited to, Northern blotting, RT PCR, in situ hybridization, Western blotting, and immunostaining.

  As used herein, when referring to a cell, cell line, cell culture, or population of cells, the term “isolated” is substantially separated from the natural source of the cell, , Cell line, cell culture, or cell population can be cultured in vitro. Furthermore, 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 the cell morphology and / or the expression of various markers. .

  The present invention may be better understood with reference to the following detailed description of preferred embodiments of the invention and the examples contained therein. However, before describing the compositions and methods according to the present invention, the present invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods. It should be understood. Of course, various modifications and changes will be apparent to practitioners skilled in this art.

Standard methods for cloning, DNA isolation, amplification, and purification, standard methods for enzymatic reactions, and various separation techniques, including DNA ligases, DNA polymerases, restriction endonucleases, etc., are known to those skilled in the art. And is generally adopted.
Many standard methods are described by Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al., 1982 Molecular Cloning . 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; Glover Ed. ) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford and UK; 1-4, Plenum Press, New York; The abbreviations and terminology used are considered standard in this field and are commonly used in professional journals such as those cited herein.

  The present invention is directed to compositions and methods comprising a basic nutrient solution, an effective amount of ErbB3 ligand, and the compositions of the present invention are substantially free of serum. The compositions and methods of the invention are useful for culturing cells, particularly differentiable cells. It will be appreciated that various components can be added to the cell culture at different points during the culture of the differentiable cells. The medium can contain components other than those described herein. However, at one point during the preparation of the medium or during the culture of differentiable cells, it is contemplated that the defined medium includes a means of basal nutrient solution and ErbB2-derived tyrosine kinase activation.

  Although the basic nutrient solution described herein is used to maintain hES cell growth and viability, in other embodiments of the invention, pluripotency or differentiation of pluripotent cells is achieved. To maintain, alternative stem cell cultures behave in substantially the same way, for example, KSR (Invitrogen), xeno-free KSR (Invitrogen), StemPro® (Invitrogen), mTeSR (Registered Trademark) l (StemCell Technologies), HEScGRO (Millipore), DMEM-based medium, and the like are exemplified, but not limited thereto.

  In another embodiment, hES cells are cultured in a defined medium as described herein in the presence and / or absence of extracellular matrix protein (ECM), such as MATRIGEL. Human embryonic stem cells cultured in the absence of ECM contain approximately 0.5-10% human serum (hS) or hS enriched fractions from 300K and / or 100K cut-off spin columns (Microcon) Includes minutes. A hES cell aggregate suspension can be produced by directly incubating hES cells in a medium containing a direct hS or hS enriched fraction. hES cells after incubation of hS or hS-enriched fraction and media container at 37 ° C. for about 30 minutes, 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 12 hours, and 24 hours Aggregate suspensions can be produced. The colony formation rate for hES cells in medium containing hS or hS enriched fractions is described in PCT / US2007 / 062755, 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 media. US Patent Application No. 11 / 8875,057, pluripotent stem cell medium, including human stem, filed Oct. 19, 2007, for culturing hES cells in defined medium substantially free of serum Methods and Compositions for FEEDER for FEEDER PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM, which are incorporated herein by reference.

  In a different embodiment, hES cell aggregate suspensions were cultured in a substantially serum free medium in the absence of exogenously added fibroblast growth factor (FGF). This is a Tromson et al. US Pat. No. 7,005,252 medium without serum but requires culturing hES cells in a medium containing exogenously added growth factors such as FGF. It distinguishes from the invention to do.

  Cellular regulation can act through transduction of extracellular signals across the membrane leading to regulation of biochemical pathways within the cell. Protein phosphorylation represents a process by 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 have an important role in the development of many pathologies in humans, including diabetes and carcinomas, and have been reported to be associated with various congenital syndromes. Serine threonine kinases (eg, Rho-kinase) are a class of enzymes that, if inhibited, treat such human diseases, including diabetes, carcinomas, various inflammatory cardiovascular diseases, and AIDS. Can be related to Most of the inhibitors that have been identified or designed so far act at the ATP binding site. Such ATP antagonists have shown the ability to target more poorly conserved regions of the ATP binding site.

The Rho-kinase family of small GTP-binding proteins includes at least 10 members including Rho A-E 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 have the same amino acid sequence and appear to have similar intracellular targets. Rho-kinase operates as the primary downstream mediator of Rho and 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 a putative pleckstrin-homology (PH) domain in the C-terminal region. The ROCK Rho-binding domain is localized to the C-terminal part of the coiled-coil region, and the Rho GTP-binding form results in enhanced kinase activity. The Rho / Rho kinase-mediated pathway is initiated by many agonists including angiotensin II, serotonin, thrombin, endothelin-1, norepinephrine, platelet-derived growth factor, ATP / ADP, and extracellular nucleotides, and urotensin II. Play an important role in communication. In regulating the target agonist / substrate, Rho-kinase plays an important role in various cellular functions including smooth muscle contraction, actin cytoskeletal tissue, cell adhesion, automotility, and gene expression. In addition, due to the role of Rho-kinase proteins in mediating many cellular functions that are perceived to be associated with the pathogenesis of atherosclerosis, inhibitors of this kinase appear to advance atherosclerosis. It may be useful in the treatment or prevention of various arteriosclerotic cardiovascular diseases associated with endothelial contraction and increased endothelial permeability. Thus, in other embodiments of the invention, agents that promote and / or support cell survival may be selected from various cell culture media, such as the Rho-kinase inhibitor Y-27632, Fasudil, and H-1152P and 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 the like, especially isolated pancreatic endoderm and pancreatic stem cell population Function by promoting reassociation of cultured hES cells or cultures derived from hES. Increased survival of hES or hES-derived cells, regardless of whether the cells were produced in suspended cell aggregates or produced from adherent plate cultures, This was achieved with or without serum or feeder. The increased survival of these cell populations facilitates and improves the purification system using a cell sorter. Therefore, cell recovery was improved. The use of Rho-kinase inhibitors such as Y27632 allows for expansion of hES-derived cell types by promoting survival from single cells isolated at serial passages or cryogenic storage. 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 types such as generally epithelial cells such as lung, thymus, kidney, neuronal It can be applied to various cell types such as retinal pigment epithelial cells.

  As used herein, the term “differentiable cell” refers to a cell or collection of cells that can differentiate into at least partially mature cells, or cell differentiation, eg, fusion with other cells, or at least part. Used to describe those that can participate in the differentiation of naturally mature cells. As used herein, “partially mature cells”, “progenitor cells”, “immature cells”, “precusor cells”. ) "," Multipotent cells "and equivalent terms include, for example, final endoderm cells, PDXl negative foregut endoderm cells, PDXl positive pre-pancreatic endoderm cells It includes terminally differentiated cells such as PDX1-positive pancreatic endoderm cells and PDX1-positive pancreatic endoderm cells. All show at least one characteristic of a genetic cell phenotype, such as morphology or protein expression, of mature cells from the same organ or tissue, but can be further differentiated into at least one other cell type. For example, normal, mature hepatocytes normally express proteins such as albumin, fibrinogen, α-1-antitrypsin, prothrombin clotting factor, iron-binding globulin, and detoxifying enzymes such as cytochrome P-450. Thus, in the present invention, a “partially mature hepatocyte” can express albumin or one or more other proteins, or can begin to have the appearance or function of a normal, mature hepatocyte. it can.

  In contrast to cell aggregates produced by previously known methods that may differ in both size and shape, the cell aggregates and methods described herein exhibit a narrow size and shape distribution. Have. That is, the cell aggregate is substantially uniform in size and / or shape. Uniformity of cell aggregate size is important for differentiation performance and culture homogeneity. Assuming equal permeability, when basic mass transport analysis is applied to an aggregate, the diffusion of oxygen and nutrients to the center of a large aggregate is slower compared to the diffusion to a smaller aggregate. It is expected to be. Because differentiation of assembled embryonic stem cells into pancreatic lineage cells relies on the transient application of specific growth factors, cultures with a mixture of aggregates of different diameters are homogeneous (size-and-shape) cells Compared to aggregates, it tends to be a non-synchronized culture. This mixture of cell aggregates results in heterogeneity, resulting in poor differentiation performance and cannot eventually adapt itself to manufacturing, scaling up and production. The cell aggregates used herein can be of various shapes, such as spheres, cylinders (desirably having equal height and diameter), or rods. While other shapes of aggregates can be used in one embodiment of the present invention, it is generally desirable for the cell aggregates to be spherical or cylindrical. In another embodiment, the cell aggregate is a sphere and is substantially constant in size and shape. For example, if the cell aggregates differ in size or are not uniform, it will be difficult to reliably manufacture and perform a large scale-up process of cells. Thus, as used herein, the phrase “substantially constant” or “substantially constant in size and shape” or equivalent expression thereof refers to the spread in uniformity of the aggregate. , It is about 20% or less. In another embodiment, the spread in uniformity of the aggregate is no more than about 15%, no more than 10%, or no more than 5%.

  The exact number of cells per population is not important, but the size of each population (and consequently the number of cells per population) is limited by the ability of oxygen and nutrients to diffuse into the central cell, and this number It will be appreciated by those skilled in the art that depends on the cell type and the nutritional demand of this cell type. A cell aggregate can contain a minimal number of cells per population (eg, 2 or 3 cells) or can contain hundreds or thousands of cells per population. Cell aggregates typically contain hundreds to thousands of cells per collection. For purposes of the present invention, cell aggregates typically have a size of about 50 microns to about 600 microns, but vary with cell type, and the size may be 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 from about 75 to 200 microns, desirably from about 100 to 150 microns. In contrast, aggregates of cylindrical or non-spherical cells that can be obtained in suspension have unequal diameters based on minor and major axes (eg, X, Y, and Z axes). . These non-spherical cell aggregates tend to be large in size and about 500 to 600 microns in diameter and height. However, if they have not differentiated, once the differentiation is initiated by the methods described herein, these non-spherical hES cell aggregates become spheres. Non-spherical cell populations include, but are not limited to, cylindrical and cube-like cell aggregates, but the size and shape are still constant.

  Many cell types can be used to form the cell aggregates described herein. In general, depending on the three-dimensional structure being designed (eg, various organ structures including pancreas, liver, lung, kidney, heart, bladder, blood vessels, and the like), the choice of cell type will vary. For example, if the three-dimensional structure is the pancreas, the cell aggregate is one or more cell types normally found in the pancreas (eg, endocrine cells somatostatin type cells such as insulin, glucagon, ghrelin, etc., endothelial cells, smooth muscle cells) Etc.) would be beneficial. One skilled in the art can select an 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, striated and smooth muscle cells), nervous system cells (eg, glue Lining cells, dendrites, and neurons), connective tissue (bone, cartilage, osteogenic and chondrogenic cells, and lymphoid tissue), parenchymal cells, epithelial cells (cavities, vessels or channels) Endothelial cells), exocrine epithelial cells, epithelial absorption cells, keratinized epithelial cells (eg, keratinocytes and corneal epithelial cells), extracellular matrix secreting cells, mucosal epithelial cells, kidney epithelial cells, lung epithelial cells, breast epithelium Cells, and the like, and undifferentiated cells (eg, embryonic cells, stem cells, other progenitor cells, etc.).

  The cell aggregates described herein may be homo cell aggregates or heterogeneous cell aggregates. As used herein, a “homo cell”, “single cell” cell aggregate or equivalent expression thereof indicates a plurality of cell aggregates in suspension. Here, each cell aggregate substantially includes a plurality of living cells of a single cell. For example, hES cell aggregates produced by the methods herein can be substantially homologous, can consist essentially of pluripotent hES cells, and can be substantial from the final endodermal cells. Can consist essentially of foregut endoderm cells, can consist essentially of pancreatic endoderm cells, and can also consist of PDX1-positive pre-pancreatic endoderm cells, PDX1- Positive pancreatic endoderm cells, PDX1-positive pancreatic endoderm, pancreatic endocrine progenitor cells, pancreatic endocrine cells, and the like.

  As used herein, the term “substantially” or “essentially” refers to “de minimus” or a small amount of components or cells present in a cell assembly suspension type. For example, a cell aggregate in a suspension described herein is “essentially or substantially homogeneous”, “essentially or substantially homogenous”, "Essentially hES cells", "essentially or substantially final endodermal cells", "essentially or substantially foregut endoderm cells", "essentially Or “substantially PDX1-negative foregut endoderm cells”, “essentially or substantially PDX1-positive pre-pancreatic endoderm cells”, “essentially or substantially PDX1- Positive pancreatic endoderm or progenitor cell "," essentially or Qualitatively PDX1-positive pancreatic endoderm end cells "," essentially or substantially pancreatic endocrine precursor cells "," essentially or substantially pancreatic endocrine cells ", and It is the same thing.

  Substantially homo-cell 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 total hES-derived cells in the culture. In other words, hES-derived cell aggregate suspension cultures such as PDX1-negative foregut endoderm, PDX-positive pre-pancreatic endoderm cells, PDX1-positive pancreatic endoderm or progenitor cells PDX1-positive pancreatic end cells, pancreatic endocrine precursor 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, each cell aggregate being Is a cell aggregate comprising a plurality of cells of at least 2, 3, 4, 5, 6 or more cell types, or at least one cell type and a non-cellular component, such as an extracellular matrix (ECM) material (eg, collagen, fibronectin) , Laminin, elastin, and / or proteoglycan). It is known in the art that such ECM components can be secreted naturally by the cell, or the cell alters the expression level of ECM substances and / or cell adhesion molecules such as lectins, integrins, immunoglobulins or cadherins. It can be genetically manipulated in any suitable way. In another embodiment, during assembly generation, either the natural ECM material or any synthetic material that mimics the ECM material can be incorporated into the assembly. For example, the methods described herein for producing hES-derived cell aggregates, such as pancreatic epithelium or pancreatic endoderm cell aggregates (or stage 4 cell aggregates), can be substantially obtained from pancreatic epithelial or endodermal cells. Although, it can also consist of other non-pancreatic epithelial type cells, or endoderm progenitor cells, and pancreatic endocrine cells (eg, insulin secreting cells) with a small cell number.

  Homo- or hetero-cell aggregates produced by the suspension methods described herein are cells called embryoid bodies (EBs), unlike the cell aggregates described in the art. Not the same as an aggregate. EBs are maintained in suspension culture within ES cells when they are overgrown in monolayer culture or in undefined media, or non-derived protocols (ie random Embryoid bodies are clearly distinguished from the cell aggregates described herein, since they are cell aggregates of differentiated and undifferentiated cells that appear when differentiated through (differential differentiation). In contrast, the present invention, discussed in detail in Examples 17 and 20, shows that the hES cells are enzymatically separated in an adherent plate culture to form a single cell suspension and then multiple cells are combined. These cell aggregate suspension cultures are used for differentiation, substantially as described in d'Amour 2005 and 2006 above. The following further discusses other differences between EBs and the cell aggregates of the present invention.

  Furthermore, other methods for forming embryoid bodies (EBs) are described. As used herein, the terms “embryoid bodies”, “aggregate bodies” or equivalent expressions are used when ES cells are overgrown in monolayer culture, Or differentiated and undifferentiated when maintained in suspension culture in undefined medium or when differentiated via a non-derived protocol (ie random differentiation) towards multiple germ layer tissue Cell assembly of cells. That is, EBs are not formed from single cell suspensions of pluripotent stem cells as described herein. Also, EBs are not formed from adherent cultures of hES-derived pluripotent cells. Only by these characteristics, the embryoid body is clearly distinguished from the present invention.

An embryoid body is a mixture of different cell types, usually several germ layers, distinguishable by morphological criteria. Embryoid bodies are mostly derived from embryonic stem (ES) cells that are undifferentiated, i.e. exposed to high concentrations of serum in which undifferentiated cells do not have defined growth factors. Reference is usually made to morphological structures composed of the resulting population of cells. Under culture conditions suitable for EB formation (eg, removal of Leukemia's inhibitory elements for mouse embryonic stem cells, or other similar blocking factors), embryonic stem cells proliferate and form small cells And begin to differentiate. Initially, corresponding to about 1-4 days of differentiation for human embryonic stem cells, the cell nodules form a layer of endoderm cells on the outer layer. This is considered to be a “simple embryoid body”. Second, a “complex embryoid body” is formed corresponding to about 3-20 days post-differentiation for human embryonic stem cells. This is characterized by extensive differentiation of ectodermal and mesoderm cells and derivative tissues. As used herein, EBs includes both simple and complex EBs, unless considered different by context.
The determination as to when embryoid bodies were formed in culture of ES cells is routinely made by those skilled in the art, for example, by visual inspection of morphology. Depending on the culture conditions, a floating mass of about 20 or more cells is considered to be EBs. See, for example, Schmitt et al. (1991) Genes Dev. 5,728-740; Doetschman et al. (1985) J. MoI. Embryol. Exp. Morph. 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. USA 95, 13726. Primordial germ cells, sometimes referred to in the art as EG cells or germ cells, can form pluripotent embryonic stem cells that can induce embryoid bodies when treated with appropriate factors. See, eg, US Pat. No. 5,670,372, and Shamblet et al., Supra.

  Various methods exist for making EBs, such as spin embryoid bodies described in (2008) Nature Protocol 3 (5): 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 (manufacture) of hES cells and cells derived from hES. This is because too many processes are required before the scale-up production actually starts. For example, in the Bauwens et al. Method, hES cells must first be seeded with the reduced growth factor MATRIGEL® before the cells are selected to begin suspension culture. The time and expense of this method is cumbersome because it requires custom designed micro-patterned tissue culture plates. Furthermore, the method of Ng et al. Is not cost effective for large scale production of hES cells and hES-derived cells, which requires the use of a centrifuge to create a more uniform EB. Finally, in all these methodologies, as in the present invention, cell aggregates are not made from single cell suspensions of pluripotent stem cells.

An embryoid body is a cell aggregate made up of a number of cell types from three germ layers, unlike the cell aggregate described in the present invention, typically an undifferentiated embryonic stem cell aggregate. Cell aggregates created by exposure to non-directed differentiation signals, such as 20% fetal bovine serum. The result of this undirected methodology is a mixture of cell types that are intended to mimic normal fetal growth in vitro. This approach is useful for examining fetal growth at the basic research level, but it is mainly concerned with cell yield, population identity, population purity, batch consistency, safety, cell function, and commodity costs However, it is not applicable to all large-scale cell therapy manufacturing processes. Moreover, regardless of any enrichment strategy used to purify a given cell type from embryoid bodies, the differentiation protocol does not provide a direct approach to generate a large population of single cell types. Following that, the pollutant population is always dominant and will prevent any attempt to refine the particular population. All previous work on creating and differentiating embryonic stem cell aggregates has one or more of the following components in their methodology:
1) Use of mice rather than human embryonic stem cells 2) Forced assembly protocol that relies on centrifugation to collect cells rather than normal cell adhesion process 3) Assembly of cell masses under static conditions 4) Non-single Aggregate formation 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 all embryoid body and germ layer cell types .
According to our knowledge, the only study that does not use 15-20% FCS to differentiate embryoid bodies is that cell aggregates are formed in forced aggregates, and then the aggregates are immediately A protocol is described in which germ layers are differentiated using appropriate media (Ng et al., Blood. 2005 106 (5): 1601). However, this work is irrelevant to the present invention because, after 10-12 days of static assembly, researchers have transferred embryoid bodies to non-assembled adherent cultures. In contrast to conventional work, the present invention 1) isolates human embryonic stem cells into single cells and rotational cultures at optimized shear rates to improve control of assembly diameter and cell survival The formation of aggregates by
2) Next, directly differentiate ES cell aggregates into final endoderm, differentiate into foregut endoderm, differentiate into pre-pancreatic foregut endoderm, then to pancreatic endoderm and finally to pancreatic endocrine cells Differentiate.
This differentiation protocol generates a final endoderm and pancreatic lineage population with high efficiency and minimal contamination 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, which are a mixture of differentiated and undifferentiated cells, typically consisting of cells from several germ layers and undergoing random differentiation, the cell assembly described herein is: Essentially or substantially homo-cells, present as a population of pluripotent, bipotent, or unipotent type cells, such as embryonic cells, final endoderm, foregut endoderm PDX1-positive pancreatic endoderm, pancreatic endocrine cells, and the like.

  The present invention uses a process that is reproducibly and easily applicable to large-scale manufacturing, by providing an economically efficient manufacturing process or method that can produce cell aggregates of substantially constant size and shape. To solve the above problems. In one particular embodiment, differentiable cells are expanded in suspension culture using the cell culture medium of the present invention. In another specific embodiment, the differentiable cells are retained in the suspension and expanded. That is, they remain undifferentiated or are prevented from further differentiation. In the art, when the term “expand” is used in connection with cell growth, it refers to cell growth and an increase in cell number, preferably an increase in viable cell number. In a specific embodiment, the cells are expanded by culturing in culture suspension for about 1 day, ie, about 24 hours or more. In more specific embodiments, 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. To expand.

The differentiation culture conditions and hES-derived cell types described herein are substantially the same as those described in d'Amour et al., 2006, supra. d'Amour et al., 2006 describes a 5-step differentiation protocol:
Stage 1 (substantially final endoderm production)
Stage 2 (substantially PDX1-negative foregut endoderm production)
Stage 3 (substantially PDX1-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 types can be produced by the suspension method described herein.

  As used herein, “final endoderm (DE)” refers to multipotent endoderm lineage cells that can differentiate into the intestinal tract or organ cells derived from the intestinal tract. According to certain embodiments, the final endoderm cell is a mammalian cell. And in a preferred embodiment, the final endodermal cell is a human cell. In some embodiments of the invention, the final endodermal cells are expressed 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 are the final endodermal Expressed in cells. In another embodiment, the one or more markers selected from OCT4, α-fetoprotein (AFP), Thrombomodulin®, SPARC, SOX7, and HNF4alpha are It is not significantly expressed in embryonic leaf cells. The final endoderm cell aggregate and method of production thereof is described in US patent application Ser. No. 11 / 021,618, filed Dec. 23, 2004, entitled DEFINITIVE ENDODERM. This application is incorporated herein in its entirety.

  Yet another embodiment of the invention relates to cell cultures and cell aggregates of “PDX1-negative foregut endoderm cells” and “foregut endoderm cells”, and equivalents thereof. PDXl-negative foregut endoderm cells are also multipotent, with various cells and thymus, thyroid, parathyroid, lung / bronchial, liver, pharynx, sac, part of duodenum and Eustachian tube Can result in a tissue containing. In some embodiments, the foregut endoderm cells are non-foregut endoderm cells such as SOX17, HNF1B, HNF1α, FOXAl, such as a final endoderm or PDX positive endoderm that does not significantly express these markers. Compared to increased levels of expression. PDXl-negative foregut endoderm cells express low or no PDXl, AFP, SOX7, and SOXl. PDXl-negative foregut endoderm cell aggregates and methods for producing them are disclosed in PDXI-expressing dorsal and PDXI-expressing dorsal and ventral foregut endoderm cells. ventral foregut endoderm), which is described in US patent application Ser. No. 11 / 588,693, which is incorporated herein in its entirety.

  Other embodiments of the invention relate to cell cultures of “PDX1-positive pancreatic foregut endoderm cells” and “PDX1-positive pre-pancreatic endoderm cells”, or equivalents thereof. PDX1-positive pre-pancreatic endoderm cells are pluripotent and can result in a variety of cells and / or tissues including the stomach, intestine and pancreas. In some embodiments, PDX1-positive pre-pancreatic endoderm cells have increased PDXl, HNF6, SOX9, and PROXl compared to non-pre-pancreatic endoderm cells that do not express these markers in high amounts. Expressed at level. PDX1-positive pre-pancreatic endoderm cells also express NKX6.1, PTFlA, CPA, and cMYC at low levels or not at all.

  Other embodiments of the present invention include “PDXI-positive pancreatic endoderm cells”, “PDXI-positive pancreatic progenitors”, “pancreatic epithelium” ) "," PE "or their equivalents. PDX1-positive pancreatic progenitor cells are multipotent and can result in a variety of cells in the pancreas including acinar, duct and endocrine cells. In some embodiments, PDX1-positive pancreatic progenitor cells express PDX1 and NKX6.1 at increased levels compared to non-pre-pancreatic endoderm cells that do not express these markers in large amounts. PDX1-positive pancreatic progenitor cells also express low or no PTFlA, CPA, cMYC, NGN3, PAX4, ARX, NKX2.2, INS, GCG, GHRL, SST, and PP.

  Alternatively, another embodiment of the invention relates to cell culture of “PDXI-positive pancreatic endoderm tip cells”, or equivalents thereof. In some embodiments, PDX1-positive pancreatic endoderm tip cells, like PDX1-positive pancreatic progenitor cells, express increased levels of PDXl and NKX6.1, but unlike PDX1-positive pancreatic progenitor cells, PDX1-positive pancreatic endoderm tip cells further express PTFlA, CPA and cMYC at increased levels. PDX1-positive pancreatic endoderm tip cells express low or no NGN3, PAX4, ARX and NKX2.2, INS, GCG, GHRL, SST, and PP.

  Other embodiments of the invention relate to cell cultures of “pancreatic endocrine precursor cells”, “pancreatic endocrine precursor cells” or equivalents thereof. Pancreatic endocrine progenitor cells are multipotent, resulting in mature endocrine cells including alpha, beta, delta and PP cells. In some embodiments, pancreatic endocrine precursor cells express NGN3, PAX4, ARX, and NKX2.2 at increased levels compared to other non-endocrine precursor cell types. Also, pancreatic progenitor cells express INS, GCG, GHRL, SST, and PP at low levels or not at all.

  Further embodiments of the invention relate to cell cultures of “pancreatic endocrine cells”, “pancreatic hormone secreting cells”, “pancreatic islet hormone-expressing cells”, or the like, obtained from pluripotent cells in vitro. Cells, such as alpha, beta, delta and / or PP cells or combinations thereof. Endocrine cells can be poly-hormonal or single-hormonal, for example, insulin, glucagon, ghrelin, somatostatin, and pancreatic polypeptide or combinations thereof. Thus, endocrine cells can express one or more pancreatic hormones. This has at least some of the functions of human islet cells. Islet hormone-expressing cells can be mature or immature. Mature pancreatic islet hormone-expressing cells and immature pancreatic islet hormones based on the differential expression of certain markers or based on functional capacity in vitro or in vivo, such as glucose responsiveness Expressing cells can be distinguished. Pancreatic endocrine cells also express NGN3, PAX4, ARX, and NKX2.2 at low levels or not at all.

  Compared to the final mesenchymal cells of the mesenchyme, most of the cell shapes are epithelialized. In some embodiments, the pancreatic endoderm cells are PDXI-expressing dorsal and ventral foregut endoderm cells (PDXl EXPRESSING DOSAL AND VENTRAL FOREGUT ENDODERM) filed Oct. 27, 2006. One or more markers selected from Table 3 and / or one or more markers selected from Table 4 described in US patent application Ser. No. 11 / 588,693, and on April 26, 2005 It expresses one or more markers of US patent application Ser. No. 11 / 115,868 entitled PDXI-expressing endoderm. These are incorporated herein by reference in their entirety.

  The present invention contemplates compositions and methods useful for differentiable cells regardless of their source or their plasticity. In this specification, as is the case in the art, cell “forming” is used in a general sense. That is, cell formability refers to the ability of a cell to differentiate into a particular cell type found in tissues or organs from the fetus, fetus or grown organism. The more “former” a cell is, the more tissue it may be able to differentiate. “Multipotent cells” include cells and their progeny. They differentiate into, give rise to, and / or several if not all, pluripotent, multipotent, oligopotent, and unipotent cells Resulting in mature or partially mature cell types found in organs from fetuses, fetuses or grown organisms. “Multipotent cells” include cells and their progeny. They differentiate into or yield multipotent, oligopotent, and unipotent cells, and / or mature or partially mature cell types are limited to specific tissues, organs or organ systems Except where the case results in one or more mature or partially mature cell types. For example, multipotent hematopoietic progenitor cells, and / or their progeny have the ability to differentiate into or result in one or more types of oligopotent cells such as myeloid progenitor cells and lymphoid progenitor cells, It also results in other mature cell compositions usually found in the blood. “Oligopotent cells” include cells and their progeny whose ability to differentiate into mature or partially mature cells is more limited than pluripotent cells. However, oligopotent cells still have the ability to differentiate into specific tissues, organs or organ systems of oligopotent and unipotent cells and / or one or more mature or partially mature cell types. be able to. One example of an oligopotent cell is a myeloid progenitor cell. This can result in mature or partially mature red blood cells, platelets, basophils, eosinophils, neutrophils and mononuclear cells. “Monopotent cells” are cells that have the ability to differentiate into or yield to other types of unipotent cells and / or one type of mature or partially mature cell type. Including descendants of.

  Differentiated cells as used herein can be multipotent, multipotent, oligopotent, or unipotent. In certain embodiments of the invention, the differentiable cell is a multipotent differentiable cell. In a more specific embodiment, the pluripotent differentiable cells are selected from the group consisting of embryonic stem cells, ICM / superblast cells, primitive ectoderm cells, primordial germ cells, and teratocarcinoma cells. In one particular example, the differentiable cell is a mammalian embryonic stem cell. In a more particular embodiment, the differentiable cell is a human embryonic stem cell.

The present invention also contemplates differentiable cells from any source in an animal provided that the cells are differentiable as defined herein. For example, differentiable cells can be obtained from a more mature tissue, such as a fetus, or any primordial germ layer within it, placenta or chorionic tissue, or adult stem cells, such as lipids, bone marrow But can be obtained from, but not limited to, neural tissue, breast tissue, liver tissue, pancreatic tissue, epithelial tissue, respiratory tissue, gonad and muscle tissue. In a specific embodiment, the differentiable cells are embryonic stem cells. In another specific embodiment, the differentiable cell is an adult stem cell.
In yet another specific embodiment, the stem cells are placental or chorionic stem cells.

  The present invention contemplates the use of differentiable cells from all animals of course capable of generating differentiable cells. The animal from which the differentiable 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, equines, scorpions, and pigs.

  Any method known to those skilled in the art can be used to induce the differentiable cells of the invention. For example, human pluripotent cells can be produced using dedifferentiation and nuclear transfer methods. Furthermore, human ICM / blastodermal upper layer cells or primitive ectoderm cells used in the present invention can be induced in vivo or in vitro. Primordial ectoderm cells can be produced as cell aggregates in adherent or suspension culture, as described in WO 99/53021. In addition, human pluripotent cells can be passaged by any method known to those skilled in the art, such as manual passage methods or bulk passage methods such as enzymatic or non-enzymatic passage methods. Can be used.

  In some embodiments, when embryonic stem cells are utilized, the embryonic stem cells have a normal karyotype, while in other embodiments, the embryonic stem cells have an abnormal karyotype. In one embodiment, the majority of embryonic stem cells have a normal karyotype. Normal 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 of the middle stage of mitosis examined It is contemplated to exhibit a karyotype.

  In another embodiment, the majority of embryonic 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 of the middle stage of mitosis examined It is contemplated to exhibit a karyotype. In certain embodiments, the abnormal karyotype is evident after the cells have been subcultured 5 or more, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 or more. In one specific embodiment, the abnormal karyotype comprises at least one autosomal trisomy selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17. In another embodiment, the abnormal karyotype comprises more than one autosomal trisomy, wherein at least one of the more than one autosome is selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17. . In one embodiment, the autosome is chromosome 12 or chromosome 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 envisioned that chromosomal translocations may occur, and such translocations are encompassed within the term “abnormal karyotype”. In addition, combinations of the above chromosomal abnormalities and other chromosomal abnormalities are included in the present invention.

  The compositions and methods of the present invention include a basic nutrient solution. As used herein, a basic nutrient solution is a mixture of certain bulk inorganic ions essential for normal cellular metabolism and a salt that provides water to the cell, A salt mixture that maintains a extracellular osmotic balance, provides carbohydrates as an energy source, and provides a buffering system for maintaining the medium within a physiological pH range. Examples of basic nutrient solutions include Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPM11640, Hams F-10 (Ham's F-10), Ham's F-12, Alpha-Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscob These include, but are not limited to, Iscove's Modified Dulbecco's Medium, and mixtures thereof. In one specific example, the basic nutrient solution is an approximately 50:50 mixture of DMEM and Hams F12.

It is contemplated that the composition further comprises 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, sulfuric acid, iron, cadmium, cobalt, chromium, germanium, sodium, potassium, calcium, phosphate and magnesium. Specific examples of the compound containing a trace element 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 , selenite-2Na, selenomethionone, SnCl ₂ , ZnSO ₄ , ZrOCl ₂ , mixtures thereof, and salts thereof. It is not limited to. When selenium, selenite or selenomethionone is present, it is present at a concentration of about 0.002 to about 0.02 mg / L. Furthermore, 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 / glutamax, 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 salt dihydrate, and L-valine. In some 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 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 present invention can include other ingredients such as serum albumin, iron binding globulin, L-glutamine, lipids, antibiotics, beta mercaptoethanol, vitamins, minerals, ATP, and the like. Can exist. Examples of vitamins that can be present include 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, tocotrienol, K ₁ and K ₂ . One skilled in the art can determine the optimum concentration for use in a particular culture, such as minerals, vitamins, ATP, lipids, essential fatty acids and the like. For example, the supplement concentration can be from about 0.001 microM to about 1 mM or more. Specific examples of concentrations at which supplements can be provided are about 0.005 microM, 0.01 microM, 0.05 microM, 0.1 microM, 0.5 microM, 1.0 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, 100 micro M, etc. It is not limited to these. In one specific embodiment, the compositions and methods of the invention include vitamin B ₆ and glutamine. In another specific embodiment, the compositions and methods of the present invention comprise vitamin C and iron supplements. In another specific embodiment, the compositions and methods of the present invention include 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 present invention comprise tocotrienol and beta mercaptoethanol. In another specific embodiment, the compositions and methods of the present invention comprise glutamine and ATP. In another specific embodiment, the compositions and methods of the present 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 present invention, alpha-linolenic acid, and B _2.

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

  The compositions and methods of the present invention include means for stimulating ErbB2 tyrosine kinase activity in differentiable cells. In a specific embodiment of the invention, the compositions and methods of the invention comprise 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 differentiable 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 heterodimeric receptor. The one 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), acetylcholine receptor Including, but not limited to, origin action (ARIA), glial growth factor 2 (GGF2), and sensory and motor neuron-derived factor (SMDF), neuregulin-2; NRG2-β Non-limiting neuregulin-2 splice variants and isoforms; epiregulin; biregulin.

  In one embodiment, the means for stimulating ErbB2-derived tyrosine kinase activity is neuregulin-1, heregulin-β (HEREGulin-β: HRG-β), heregulin-α (HRG-α), neudifferentiation factor (NDF). ), Acetylcholine receptor-derived activator (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), neuregulin-2, neuregulin-2β (NRG2-β) And at least one ErbB3 ligand selected from the group consisting of epiregulin, biregulin, and variants thereof, functional fragments. In another specific embodiment, the compositions and methods of the present invention provide more than one means of stimulating ErbB2-derived tyrosine kinase activity, such as, but not limited to, using two or more ErbB3 ligands. Including.

  In further specific embodiments, in the compositions and methods of the invention, the ErbB3 ligand is HRG-β, or a variant, functional fragment thereof. In one embodiment, the functional protein of the medium-added protein, polypeptide, variant or derivative thereof is the same as the cell type to be cultured. For example, when mouse embryonic stem cells are cultured, HRG-β having the same amino acid sequence as the mus musculs HRG-β sequence can be used as an additive in the culture and is considered to be “same species”. In other embodiments, the species derived from the biological additive will be different from the cells being 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 considered to be a “different species”.

  As used herein, “functional fragment” refers to a full-length polypeptide fragment or splice variant that produces a similar physiological or cellular effect as a full-length polypeptide. . The biological effect of a functional fragment need not be in the same range or strength 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 an ErbB2-derived tyrosine kinase.

  As used herein, the term “variant” includes chimeric or fusion polypeptides, homologues, analogs, and orthologs or paralogs. Further, a referenced protein or polypeptide variant is a protein or polypeptide that is at least about 80% identical in amino acid sequence to the referenced protein or polypeptide. In specific embodiments, the variant is at least about 85%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% the same as the comparison protein or polypeptide. When the term “corresponding” is used herein in the context of sequence alignment, it is intended to mean the stated position within the referenced protein or polypeptide. For example, wild type human or mouse neuregulin-1 and the position in the referenced protein or polypeptide in the modified protein or polypeptide. Therefore, 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 a given position in the amino acid sequence of the referenced protein or polypeptide Is a sequence that is aligned with the position of the referenced sequence and does not have to be in the exact same position as the reference sequence. A method for aligning sequences to determine corresponding amino acids between sequences is described below.

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

  As used herein, the term “identity” is a measure of the identity of a nucleotide or amino acid sequence compared to a referenced nucleotide or amino acid sequence. In general, the sequences are arranged so that the highest order match is obtained. “Identity” itself has a recognized meaning in the art and can be calculated by known techniques. (See, eg, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects ), Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data (Part I), Griffin, AM, 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 (Sequence Analysis Primer), Gribskov, M.and Devereux, J., eds, M Stockton Press, New York (1991)). Although there are several ways to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is known to those skilled in the art (Carillo, H. & Lipton, D. et al. , Siam J Applied Math 48: 1073 (1988)). Commonly used methods for determining identity or similarity between two sequences are not limited to these, but include Guide to Huge Computers, Martin J. et al. Bishop, ed. , Academic Press, San Diego (1994) and Carillo, H .; & Lipton, D.C. , Siam J Applied Math 48: 1073 (1988). A computer program can include methods and algorithms for calculating identity and similarity. Examples of computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (i): 387 (1984)), BLASTP, ExPASy5 BLASTN, FASTA (Atschul, SF, et al., J Molec Biol 215: 403 (1990)) and FASTDB. Examples of methods for determining identity and similarity can be found in Michaels, G. et al. and Garian, R.A. , Current Protocols 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 according to 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 given as a percentage. The parameters used for FASTDB alignment of amino acid sequences to calculate percent identity are, but are not limited to: Matrix = PAM, k-tuple = 2, mismatch penalty = 1, joining penalty = 20, randomization group length = 0, cut-off score = 1, gap penalty = 5, gap size penalty 0.05, win Dorsize = 500, or the length of the target amino sequence, whichever is shorter.

  If the sequence of interest is shorter or longer than the sequence of interest for N-terminal or C-terminal addition or chromosomal deletion, manually, regardless of whether it is for internal addition or chromosomal deletion It can be corrected. This is because when calculating for percent identity, the FASTDB program does not calculate the N-terminal and C-terminal truncations or additions of the subject sequence. For sequences that are truncated at the 5 ′ or 3 ′ end with respect to the sequence of interest, the percent identity is the base sequence of the subject sequence that is N-terminal and C-terminal to the unmatched / aligned subject sequence. Is corrected as a percentage of the total bases of the subject sequence. The result of the FASTDB sequence alignment determines the matching / alignment. The percentage of alignment is subtracted from the percent identity calculated with the FASTDB program above using the specific parameters to obtain the final percent identity score. This modified score, as well as percent identity, can be used to determine how alignments “correspond” to each other. For the purpose of manually adjusting the percent identity score, the residue of the query (subject) sequence extending 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 a 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. Chromosomal deletions occur at the N-terminus of the subject sequence. Thus, the FASTDB sequence does not show a match / alignment of the first 10 residues at the N-terminus. 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), from the percent identity score calculated by the FASTDB program 10% is drawn. If the remaining 90 residues are perfectly aligned, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 subject sequence. At this time, a chromosomal deletion is an internal defect that has no residues at the N- or C-terminus of the subject sequence, does not match the subject, or is not aligned.
In this case, the percent 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 a portion of a member of a reference polypeptide that is operatively linked to a second different polypeptide. The second polypeptide has an amino acid sequence that corresponds to a polypeptide that is not substantially the same as the reference polypeptide, wherein the polypeptide is derived from the same or different organism. With respect to fusion polypeptides, the term “effectively links” is intended to cause the reference polypeptide and the second polypeptide to fuse with each other to achieve the expected function that occurs in the sequence 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 certain embodiments, the fusion polypeptide is a GST-IGF-1 fusion polypeptide in which an IGF-1 sequence is fused to the C-terminus of the GST sequence. Such fusion polypeptides can facilitate the purification of the recombinant polypeptide. In another embodiment, the fusion polypeptide can include a heterologous signal sequence at the N-terminus. In certain host cells (eg, mammalian host cells), polypeptide expression and / or secretion can be increased through the use of heterologous signal sequences.

  In addition to fragments and fusion polypeptides, the present invention includes homologs and analogs of naturally occurring polypeptides. “Homologue” 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 antagonists as defined below. The term “homolog” further encompasses nucleic acid molecules that differ from a reference nucleotide sequence due to the degeneracy of the genetic code, and thus encode the same polypeptide encoded by the reference nucleotide sequence. As used herein, “naturally occurring” refers to a naturally occurring nucleic acid or amino acid sequence.

  An agonist of a polypeptide can retain substantially the same or a subset of the biological activity of the polypeptide. Polypeptide antagonists can inhibit one or more of the activities that naturally occur from polypeptides.

  In a more specific embodiment of the compositions and methods of the invention, the ErbB3 ligand is HRG-β, a variant or a functional fragment thereof. In addition, non-limiting examples of ErbB3 ligands are disclosed below. U.S. Pat. 6,136,558, 6,387,638, and 7,063,961. These are hereby incorporated by reference.

  In general, heregulin is classified into two main types, alpha and beta, based on two mutant EGF-like domains that differ in the C-terminal part. 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 HRGs were 45% similar to heparin-binding EGF-like growth factor (HB EGF) between the 1st to 6th cysteines of the EGF-like domain, and amphiregulin ( AR) was found to be 35% identical, 32% identical to TGF-α and 27% identical to EGF.

  44 KDa New Differentiation Factor (NDF) is the rat equivalent of human HRG. Like the HRG polypeptide, NDF lacks an N-terminal signal peptide, with an EGF-like domain following an immunoglobulin (Ig) homology domain. There are currently 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 to 4 are characterized based on varying stretches between the EGF-like domain and the transmembrane region. Thus, it appears that different NDF isoforms are generated by splicing changes and can perform different tissue-specific functions. See EP 505148; WO 93/22424; and WO 94/28133. These are hereby incorporated 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 exogenous insulin or insulin substitutes” is used herein to indicate that insulin or insulin substitutes are not intentionally 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 intentionally supplied insulin or insulin substitutes. However, the compositions or methods of the present invention are not necessarily unable to step on endogenous insulin. As used herein, “endogenous insulin” indicates that cultured cells can produce insulin as their own action when cultured by the method of the present invention. Endogenous insulin may be used to indicate impurities from primary cell cultures or residual impurities from starting materials. In specific examples, the compositions and methods of the present invention are 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 Contains micro g / mL or less of insulin.

  As used herein, the term “insulin” refers to a protein, variant or fragment thereof that can bind to an insulin receptor at a normal physiological concentration and cause a signal through the insulin receptor. The term “insulin” encompasses all natural human insulin, other mammalian insulin, proteins having a polypeptide sequence that is a homologue or variant of these sequences. Furthermore, the term insulin encompasses polypeptide fragments that can bind to the insulin receptor and cause a signal through the insulin receptor. The term “insulin surrogate” also includes all zinc-containing compounds that are 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 sulfate.

  Specifically, insulin-like growth factor is not an insulin substitute contemplated by the present invention, nor is it a homologue of insulin. Accordingly, in another specific embodiment, the compositions and methods of the present invention comprise 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 present invention do not include any exogenous insulin-like growth factors (IGFs). In specific embodiments, the compositions and methods of the present invention are 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng / Contains up to mL IGF-1.

As used herein, the term “IGF-1R activator” refers to a mitogenic factor that plays a pivotal role in regulated cell growth, differentiation, and apoptosis. The effect of an activator of IGF-1R can be mediated through other receptors, but is usually mediated through IGF-1R. IGF-1R also includes cell transformation caused by oncovirus proteins and oncogene products, and the interaction is regulated by a group of specific binding proteins (IGFBPs). Furthermore, a large group of IGFBP proteases hydrolyze IGFBPs, dissociate IGFs binding, 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 analog. Non-limiting examples of IGF-1 analogs include LongR3-IGF1, Des (l-3) IGF-1, [Arg ³ ] IGF-1, [Ala ³¹ ] IFG-1, Des (2, 3) [ Ala ³¹ ] IGF-1, [LeU ²⁴ ] IGF-1, Des (2,3) [Leu ²⁴ ] IGF-1, [Leu ⁶⁰ ] IGF-1, [Ala ³¹ ] [Leu ⁶⁰ ] IGF-1, Leu ²⁴ ] [Ala ³¹ ] IGF-1, and combinations thereof. In a further embodiment, the IFG-1 analog is LongR3-IGF1. LongR3-IGFl is a recombinant analog of human insulin growth factor-1. LongR3-IGFl is about 1 ng / mL to about 1000 ng / ml, more preferably about 5 ng / ml to about 500 ng / mL, more preferably about 50 ng / ml to about 500 ng / mL, more preferably about 100 ng / ml to about It is assumed that it is 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 a TGF-β family, variant, or functional fragment thereof. As used herein, the term “TGF-β family member” or similar term used in connection with growth factors is a general term used by those skilled in the art related to the TGF-β family. Or by homology known to members of the TGF-β family or by functional similarity known to members of the TGF-β family. In a specific embodiment of the invention, a TGF-β family member, variant, or functional fragment thereof activates SMAD2 or 3 when a TGF-β family member is present. In one embodiment, the TGF-β family member is from the 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.

  When Nodal is present, it is about 0.1 ng / mL to about 2000 ng / ml, more preferably about 1 ng / ml to about 1000 ng / mL, more preferably about 10 ng / ml to about 750 ng / mL. More desirably, it is contemplated to be present at an initial concentration of about 25 ng / ml to about 500 ng / ml. When used, activin A is about 0.01 ng / mL to about 1000 ng / ml, more desirably about 0.1 ng / ml to about 100 ng / mL, more desirably about 0.1 ng / ml to about 25 ng / ml. It is contemplated that it is present at an initial concentration of mL, or desirably about 10 ng / ml. When 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 contemplated 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 comprise an activator 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 used by those of ordinary skill in the art related to the FGF-β family, or known to members of the FGF-β family. Refers to growth factors characterized by similar 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 not limited to, αFGF and FGF2. In a specific embodiment, the compositions and methods do not include exogenous FGF2. The term “exogenous FGF2” is not used herein as an indication that no fibroblast growth factor 2, ie basic FGF, is deliberately added to the composition or method of the invention. Thus, in certain embodiments of the invention, the methods and compositions of the invention do not include deliberately supplied FGF2. However, the compositions or methods of the present invention cannot necessarily include endogenous FGF2. As used herein, “endogenous FGF2” indicates that the cultured cell itself can produce FGF2 when cultured by the method of the present invention. Endogenous FGF2 may be used to indicate impurities from primary cell culture or residual impurities from starting material. As a specific example, a composition or method of the present invention comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng / mL or less of FGF2.

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

In a further embodiment, the compositions and methods of the present 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 about 10% v / v or less. In further specific embodiments, the concentration of SA is about 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 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 present invention include at least one insoluble substrate. For example, but not limited to, differentiated cells can be placed on a cell culture surface containing compounds such as polystyrene, polypropylene, and the surface can then 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 nidogen.

Accordingly, the cell culture environment and method of the present invention includes plating cells for adhesion culture. As used herein, the terms “plated” and “plating” refer to any process that allows cells to grow in adherent culture. As used herein, the term “adhesive culture” refers to a culture system in which cells are cultured on an individual surface, the individual surface being coated with an insoluble substrate, and then a substrate as exemplified below. It is coated with other surface coatings, or with any other chemical or biological material that allows the cells to grow or stabilize. The cells may or may not adhere firmly to a solid surface or substrate. Substrates for adherent culture include polyornithine, laminin, polylysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycan, polysaccharide degrading acid (PGA), polylactic acid (PLA), and One of polylactic acid glycolic acid (PLGA) or any combination thereof may be included. Furthermore, the substrate for adhesion culture can comprise a feeder layer or a matrix comprising multipotent human cells or cell cultures. As used herein, the term “extracellular matrix” encompasses (but is not limited to) the solid substrates described above and includes feeder layers or multipotent human cells or cell cultures. Includes a matrix. In one embodiment, the cells are plated on a plate coated with MATRIGEL®. In another embodiment, the cells are plated on fibronectin-coated plates. In one embodiment, when cells are plated on fibronectin, the plate is prepared with a coating of 10 microg / mL human plasma fibronectin (Invitrogen, # 33016-015), diluted with tissue grade water, and room temperature. At 2-3 hours.
In another embodiment, serum can be placed in the medium for up to 24 hours to allow cells to be plated on 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 the differentiable cells are cultured under conditions that are essentially free of feeder cells or feeder layers. As used herein, “feeder cells” are cells that grow 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 are free of feeder cells or contain a number of “de minimus” feeder cells. Indicates. The term “deminimus” refers to the number of feeder cells that have been 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 target cells in the current state of differentiation. In another embodiment, the defined medium is an unconditioned medium, which is a medium not obtained from feeder cells.

  As used herein, the term “stable” when used in connection with the differentiation state of a cell or cell culture cell, preferably means that the cell continues to grow in the medium for several generations, preferably the medium. It means that the cells in the medium continue to grow and most if not all cells are in the same differentiated state. When more stable cells divide, the division usually results in cells of the same cell type or cells of the same differentiation state. In general, stable cells or cell populations do not further differentiate or redifferentiate unless the culture conditions are changed, and the cells continue to be passaged and do not overgrow. In one embodiment, cells that are stable are capable of growing in a steady state indefinitely or for at least two or more generations. In a more specific embodiment, the cell is 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, Cells are stable for over 25 generations and over 30 generations. 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 over consecutive months. In another embodiment, the cells are stable for about 1 year or more of continuous passages. In one embodiment, the stem cells are maintained at normal passage in a defined medium 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 of BMP signaling. As used herein, “an inactivator of BMP signaling” refers to antagonism through the activity of one or more BMP proteins, or any of their upstream or downstream signal components, and any of the possible signaling pathways. It refers to the drug to be used. The one or more compounds used for the inactivate of BMP signaling can be any compound known in the art or discovered in the future. Non-limiting examples of inactivators of BMP signaling include dominant-negative, truncated BMP receptor, soluble BMP receptor, BMP receptor-Fc chimera, noggin, follistatin ( follistatin, nerve-derived factor (chordin), gremlin (gremlin), cerberus / DAN family proteins (cerberus / DAN family proteins), bentropin (ventropin), high dose activin (high dose activin), and amnionless (amnionless) .

  In certain embodiments, the compositions and methods of the present invention can include at least one hormone, cytokin, 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 differentiable cells cultured in the defined medium. Thus, for example, if human cells are cultured, the growth hormone is human growth hormone. It is also envisaged to use growth hormone from a different species than the cultured cells. Desirably, the hormone, cytokin, adipokine, and / or growth hormone is 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. Present at an initial concentration of .01 ng / ml to about 150 ng / ml.

  Examples of cytokins and adipokines that can be included in the compositions and methods of the present invention include the 4α helix bundle family of cytokins, the interleukin-1 (IL-I) family of cytokins, the IL-17 family of cytokins, And the chemokine family of cytokins, including but not limited to. Of course, the present invention contemplates members and subclasses of each of these families of cytokines, for example, CC chemokines, CXC chemokines, C chemokines and CX3C chemokines, interferons, interleukins, lymphotoxins, c -Kit ligand, granulocyte / macrophage colony stimulating factor (GM-CSF), monocyte-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, visfatin, retinol binding protein 4 ( RB 4), erythropoietin (EPO), but Suronbopoechin (THPO) are exemplified, but the invention is not limited thereto. Of course, those skilled 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 for culturing differentiable cells, the method comprising plating the differentiable cells on a cell culture surface, providing the cells with a basic nutrient solution and providing the cells with ErbB2-derived tyrosine. Providing a means for stimulating kinase activity.

  In one embodiment, at least one composition of the invention is contacted with a differentiable cell in the absence of serum or serum replacement, and in the absence of a feeder cell layer, and the cell is at least 1 month old. Maintained in a differentiated state. Pluripotency can be determined through cell characterization with respect to surface markers, transcriptional markers, karyotype, ability to differentiate into cells of three germ layers. These characterizations are well known to those skilled 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 involved. Tissue culture media can include, for example, basic nutrients such as sugars and amino acids, growth factors, antibiotics (to minimize contamination), and the like. In another embodiment, differentiable cells are cultured in suspension using the cell culture media 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 not limited to, flow hoods, incubators, and / or devices used to maintain cells in constant motion. For example, a stirrer platform, a shaker, etc. can be provided as needed. As used herein, a cell has “movement” if the cells are moving, or if the environment between them is moving relative to the cells. Once the cells are maintained in a “movement” state, in one embodiment, the movement is designed to avoid or prevent exposure of the cells to shear forces. It will be.

  Various methods of making cell aggregates are known in the art, such as the “hanging drpo” method, in which cells are placed at the bottom of a drop in an inverted drop of tissue culture. Sink, collect and shake the cell suspension in a laboratory flask. Various variations of these techniques can also be used. For example, N.I. E. Timmins et al., (2004) Angiogenesis 7, 97-103; Dai et al. (1996) Biotechnology and Bioengineering 50, 349-356; A. Foty et al. (1996) Development 122, 1611-1620; Forgacs et al., (2001) J. MoI. Biophys. 74, 2227-2234 (1998); 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; Korff et al. (2001) FASEB J. et al. 15, 447-457. All of which are hereby incorporated by reference. More recently, cell aggregates scrape micropatterned colonies into the suspension, centrifuge the microtiter plate colonies into suspension, or use a pipette to form patterned microwells. The colonies grown in (1) are removed and suspended (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 subject to synchronized directed-differentiation. Optimized as described in d'Amour et al. 2006 above. Unlike these other methods, the methods for producing cell aggregates in suspension described herein are susceptible to large scale production.

  In general, the cell culture composition of the present invention is refreshed at least once a day, but more or less can vary depending on the specific needs and circumstances of the suspension culture. In vitro, cells are grown in culture, usually in batch mode, and exposed to various media conditions. As described herein, the cells are present in the dish medium as an adherent culture or as a cell aggregate in suspension, contacting the surrounding medium; and periodically the waste medium is replaced. 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 medium 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. Additional examples include 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 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 drugs that must be frequently replaced, or from the medium for a period of time. A reflux method was adopted as a means of depleting waste. For example, US Pat. No. 5,320,963 describes a bioreactor for perfusion culture of suspension cells. US Pat. No. 5,605,822 describes a bioreactor system that uses stromal cells to provide growth factors for the growth of HSC cells in culture by reflux. U.S. Pat. No. 5,646,043 describes the growth of HSC cells in continuous cyclic perfusion containing a medium composition for the growth of HSC cells. US Pat. No. 5,155,035 describes a bioreactor for suspension culture of cells by fluid medium rotation. These are referenced and incorporated in their entirety herein.

  In general, cells cultured in suspension in the medium composition of the present invention are “split” or “passaged” approximately weekly, although the cells are subject to specific needs and suspension cultures. Depending on the environment, it can be passaged more frequently or less frequently. For example, the cells can be 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 Can be passaged at any time frame. As used herein, the term “split” or “passage” is used in its art-relevant meaning when it is used in the context of cell culture. That is, a 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 process and methods of cell culture passage, which is not necessarily the use of enzymatic or non-enzymatic methods that can be used to separate cells that assemble together during their growth expansion. including.

In some cases, some cell death may occur in cells that have just been passaged (in suspension or adherent culture). In one embodiment, differentiable cells can be “recovered” from passage by delaying cell media refresh for more than 24 hours. Thereafter, the cell medium can be changed more frequently. In another embodiment, the cell culture medium can further comprise a cell death inhibitor. For example, recently Wantanabe et al. Discloses the use of a Rho-associated kinase inhibitor, Y27632, to protect human embryonic stem cells after dissociation. Wantanabe, K.M. , Et al., Nat. See Biotechnol, 25 (6): 681-686 (2007). This is referenced and incorporated herein.
In additional embodiments, the cell culture medium can include caspase inhibitors, growth factors or other nutrients to prevent or attenuate cell death immediately after passage. Specific examples of substances that can be used include HA1077, Dihydrochloride, Hydroxyfasudil, Rho kinase inhibitor, Rho-kinase inhibitor II, Rho kinase inhibitor III, kinase inhibitor IV, and Y27632. However, it is not limited to these. All 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 culture medium after the cell has recovered from the passage process. . In additional embodiments, undifferentiated embryonic stem cells can be effectively assembled in standard base media and do not require Y27632 or other involvement to maintain viability during dissociation and assembly. .

  In additional embodiments, the compositions and methods of the invention can also include the presence or use of a surface active compound. In one particular embodiment, the compositions and methods of the present invention include at least one surfactant in suspension culture. Surfactant compounds are well known in the art and are generally amphiphilic. In a specific embodiment, the present invention includes the use of at least one surfactant that is anionic, cationic, nonionic or zwitterionic. Determining the concentration of surfactant 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 surfactant compounds in cell culture methods of HeLa cells and human amniotic cells. Owen et al. 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 materials: sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfates; sodium laureth sulfate ( SLES), alkylbenzene sulfonate esters, soaps or fatty acid salts, cetyltrimethylammonium bromine (CTAB) (hexadecyltrimethylammonium bromide), and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), dodecylbetaine, dodecyldimethylamine oxide, cocamidopropylbetaine, yacyanfoglycinate, alkyl Poly (ethylene oxide), polyethylene oxide and polypropylene oxide copolymers such as Pluronic F68, alkyl polyglycosides such as octyl glucoside, decyl maltoside, fatty alcohol, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA and cocamide TEA And / or polyoxyethylene-sorbitan monolaurate (tween), but is not limited thereto.

  Embodiments described herein provide large scale manufacturing that maintains a low shear environment to maintain the cell concentration of the system, minimize fluid shear stress, grow and / or differentiate hES cells. Provide a way for. In particular, the present invention provides a low in eukaryotic cell production scale-up system by culturing cell suspensions in 60 mm dishes, 6 well plates, rotating bottles, bioreactors (eg, spinner flasks), containers, and the like. A method for maintaining a shear environment is provided. Alternatively, a continuous perfusion system for culturing cells can agitate or move in a bioreactor or vessel for cell suspension, oxidation, and provision of fresh nutrients, i.e., for growth and / or differentiation. I need. In order to obtain a cell suspension, a bioreactor vessel typically uses one or more movable mechanical agitation devices that are a source of shear stress.

In order to maintain cell growth and viability, it is important to establish and maintain a constant and optimized stirring shear rate. For example, increased shear rate is detrimental in the following ways:
(1); Excessive shear increases energy consumption.
(2); Excessive shearing interferes with diffusion at the cell membrane surface.
(3); Excessive shear may deprive a compound of physiological activity.
(4); Excessive shear deforms the cell membrane beyond the threshold tension of failure leading to cell lysis.
Therefore, it is desirable to maintain a shear within the range of 5 to 500 sec- ¹ , depending on the cell aggregate diameter, single cell dissociation and the sensitivity of the particular cell line to shear. An example of shear rate in a configuration useful in the method of the present invention is shown in Example 17 for a collective diameter of 100-200 microns in diameter in a 6-well dish with a rotational speed between 60-140 rpm. . These values estimate the time average shear stress that occurs in the bulk fluid during rotation. However, the shear stress at the vessel wall is expected to be higher due to boundary effects. Using the Ley et al method described above, wall shear stress is calculated for rotational speeds from 60 rpm to 140 rpm and is shown in Examples 17-19.

Other examples of means or devices for generating gently stirred cell suspensions exist and are well known to those skilled 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 swing back and forth or rotate, or Examples thereof include a combination thereof for producing a cell suspension. In the method of the present invention, a rotating platform is an exemplary means of dispersing cells in a 6-well plate and generates a shear rate of less than 400 sec- ¹ . Regardless of the stirrer type or mechanism to generate the agitated fluid suspension, the estimated time averaged shear rate and shear stress in the bulk fluid is a standard to which all fluid mixing devices are associated. Provide a visualization element. The flow conditions in the device can vary depending on their profile and the degree of laminar or turbulent flow, and the calculation of shear is the basis for equalizing the flow in the device generated by mixing by different mechanisms. provide. For example, in a 125 mL spinner flask with 4 cm impeller diameter, 6.4 cm container width, 90 degree impeller angle, and 0.1 cm impeller width, in a 6 well dish with 5 mL medium rotating at 100 rpm, For a population of lOOμm in diameter, the same time average shear rate and shear stress are generated in the bulk fluid.

  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 method of the invention can be used to maintain the low shear environment of the scale-up manufacturing system for 26, 27, 28, 29, 30 days, 40 days or more, 50 days or more. Exemplary operating times are 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 before and after contact with the defined medium of the present invention. Non-limiting examples of enzymatic dissociation methods include the use of proteases such as trypsin, collagenase, dispase, ACCUTASE®. In one embodiment, ACCUTASE® is used for passage of contacted cells. When enzymatic passage methods are used, the resulting culture can include singlets, doublets, triplets, and cell clumps, which were used. The size varies depending on the type of enzyme. A non-limiting example of a non-enzymatic dissociation method is a 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 selection of the passage method is influenced by the choice of extracellular matrix, but can be readily determined by one skilled in the art.

  In one specific embodiment, a method of culturing differentiable cells provides a dissociation solution of a layer of differentiable cells contained in a culture chamber prior to dissociation dissociating the cell layer into single cells. Including doing. After dissociation, the single cells are placed in a stem cell culture medium and a new tissue culture chamber; where the stem cell culture medium contains basal nutrient solution and ErbB3 ligand. Once cultured, single stem cells are subjected to conditions that allow single cell growth and division. In another specific embodiment, the method of culturing differentiable cells comprises providing a dissociation solution to the differentiable cell population contained in the previous culture chamber, wherein the dissociation solution is provided. Breaks a population of cells into single cells or into smaller populations.

  The disaggregation solution used in the method of the present invention should be a disaggregation solution that can disaggregate or disaggregate cells into single cells without causing large-scale acute toxicity to the cells. it can. Examples of disaggregation 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 every cell in the confluent layer or suspension be disaggregated into a single cell, provided that at least some of the individual cells can be disaggregated and re-cultured.

At the beginning of culture or after passage, cells that can differentiate at any density can be seeded, and there can be one cell in the culture chamber. Various factors can adjust the cell concentration of the seeded cells. For example, adhesion culture, use of suspension culture, specific formulation of the cultured cell culture medium used, growth conditions, and envisaged 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 with good cell survival. Any value between these values can be employed.

  In addition to the above, as used herein, the term “cell concentration manipulation”, “engineered cell concentration” or equivalent expression thereof, means that cell density in a manufacturing process or system is manipulated and proliferated. Refers to producing a sex or differentiated hES cell culture. Such cell concentrations are those under which nutrients such as vitamins, minerals, amino acids, metabolites, or environmental conditions such as oxygen partial pressure supplied to the system are sufficient to maintain cell viability. Say. Alternatively, such cell concentrations refer to those under which waste can be removed from the system at a rate that can maintain cell viability. One skilled in the art can readily determine such cell concentrations.

The working cell concentration that can be maintained is from at least about 0.5 × 10 ⁶ cells / mL. A typical scale-up system working cell concentration is between about 0.5 × 10 ⁶ cells / mL and about 25 × 10 ⁶ cells / mL. Exemplary concentrations are between about 2.5 × 10 ⁶ cells / ml, 22 × 10 ⁶ cells / mL and up to 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 permissive cell viability levels are recognized by those skilled in the art and can be determined by techniques well known to those skilled in the art. For example, in batch, fed-batch and continuously fed configurations, the cell concentration may be between about 0.5 × 10 ⁶ cells / mL and 15 × 10 ⁶ cells / mL.

  Also, differentiable cells can be utilized in the screen for molecules or elements that affect their formation or other properties. For example, differentiable cells can be used to identify agents that cause apoptosis, differentiation, or differentiated lineage generated from proliferating or differentiable cells.

  Since the compositions and methods of the present invention allow for single cell passage, differentiable cells can be successfully cultured in, for example, but not limited to, high-throughput settings such as 96-well plates or 384-well plates. It was. FIG. 16 shows the morphology and alkaline phosphatase staining of BG02 cells cultured in DC-HAIF in both 96 and 384 well plates using the method of the present invention. Briefly, hESCs cells passaged using ACCUTASE (R) and plated in 96-well and 384-well plates produced colonies similar to those observed using other culture ridges. Showed the rate. In addition, the cells formed colonies and expanded successfully in a smaller environment for 5 days. These smaller cultures remained morphologically undifferentiated and stained uniformly positive with alkaline phosphatase, a marker for undifferentiated cells. In addition, ACEA Biosciences, Inc. A 96-well culture device that provides real-time impedance measurements that can measure cell proliferation and viability by using the RT-CES® method from www.acebio.com I was able to grow hESCs. Such an approach would allow identification and quantification without subtle or immediate effect labels in differentiable cells. It will also enable real-time measurement of proliferation, apoptosis and morphological changes.

  The compositions and methods of the present invention may actually include any combination of line segments described above or elsewhere herein. However, the compositions and methods of the present invention include a basic nutrient solution and means for stimulating ErbB2-derived tyrosine kinase activity. Those skilled in the art will appreciate that the components of the compositions and methods of the present invention will vary depending on the protocol design. Accordingly, one embodiment of the invention relates to culturing differentiable cells in 96 well plates and / or 384 well plates. Using the compositions and methods of the present invention, the cell culture chamber, i.e., culture ridge, is not limited to a particular size. Thus, the methods described herein are in no way limited by the specific culture chamber dimensions and / or means and apparatus for generating cell aggregates.

  The compositions and methods described herein have several useful features. For example, the compositions and methods described herein are useful for modeling the early stages of human development. Furthermore, the compositions and methods described herein are useful for therapeutic intervention in diseases, such as by the development of pure tissue or cell types, such as neurodegenerative disorders, diabetes or renal failure. Useful for.

  Cell types that differentiate from differentiable cells include, but are not limited to, many of the research and development including drug discovery, drug development and testing, toxicology, therapeutic purposes, and production of cells for basic science research. Have uses in the field. These cell types express molecules of interest in various research fields. These include molecules known to be necessary for the function of various cell types described in standard reference texts. 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 differentiable cells of the invention can be differentiated through contact with a cell differentiation environment. As used herein, the term “cell differentiation environment” refers to a cell that is induced such that differentiation of a differentiable cell is induced or human cell culture is enriched in differentiated cells. Culture conditions are shown. Desirably, differentiated cell lineage caused by growth factors will be homogeneous. The term “homogeneity” 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 desired cell lineage.

  Cell differentiation media or environments can be utilized to differentiate, in part, terminally or reversibly, the differentiable cells of the invention. According to the present invention, the culture medium of the cell differentiation environment is composed of various components such as KODMEM medium (Knockout Dulbecco's Modified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetal bovine serum), FGF2 (fibroblast growth factor 2). ), KSR or hLIF (human leukemia inhibitory factor). The cell differentiation environment can also include supplements such as L-glutamine, NEAA (non-essential amino acids), 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); transforming growth factor (TGF) / bone-forming protein (BMP) / growth and differentiation factors ( GDF) family antagonists such as, but not limited to, noggin, follistatin, cordin, gremlin, cerberus / DAN family proteins, bentpins, high dose activins, and amnionless; variants or their mechanisms Fragments. A TGF / BMP / GDF antagonist can also be added in the form of a TGF / BMP / GDF receptor-Fc chimera. Other elements that can be added include, but are not limited to, molecules that can activate or inactivate signaling through the Notch receptor family, including but not limited to delta-like ( It includes Delta-like and Jagged family proteins, and Notch processing, or inhibitors of cleavage, or variants and functional fragments thereof. Other growth factors include insulin-like growth factor family (IGF), insulin, wingless related (WNT) factor family, hedgehog factor family (hedgehog factor family) factor family), variants or functional fragments thereof. To promote the growth and survival of endoderm stem / progenitor cells, endoderm stem / progenitor cells, mesodermal stem / progenitor cells, or final endoderm stem / progenitor cells, and the origin of these progenitor cells Additional elements can be added to promote body survival and differentiation.

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

  Progression of differentiable cells into the desired cell lineage, or maintenance in an undifferentiated state, quantifies the expression of the marker gene signature of the desired cell lineage, as well as the lack of marker gene signature of the differentiable cell type. Can be monitored. One method for quantifying the gene expression of such marker genes is the use of quantitative PCR (Q-PCR). Methods for 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 marker gene of interest.

In certain embodiments, screening methods include methods that identify compounds capable of modulating pluripotency, proliferation and / or differentiation of differentiable cells, including:
(A); providing a differentiable cell;
(B) culturing the cells in a composition comprising a basic nutrient solution and an ErbB3 ligand and substantially free of serum;
(C) contacting the cell with the test compound; and determining whether an increase or decrease in pluripotency, proliferation, and / or differentiation occurs in the cell contacted with the compound.
Where this increase is occurring, this indicates that the compound modulates pluripotency, proliferation, and / or differentiation. In certain embodiments, the ErbB3 ligand is HRG-β. In other embodiments, the ErbB3 ligand can be replaced with a test compound to determine the effect of the test compound. For example, the pluripotency, proliferation, and / or differentiation effects that occur with a test compound are compared to the pluripotency, proliferation, and / or differentiation effects that occur with an ErbB3 ligand, and the test compound to a differentiable cell Can determine the effect. It is envisioned that all compositions described herein can be used in the screening methods of the invention.

  In yet another embodiment, the ErbB3 ligand (ErbB2-derived tyrosine kinase activity) is cultured in the absence of an ErbB3 ligand (ErbB2-derived tyrosine kinase activity) and has an effect on the cells. 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 lineage. Alternatively, a composition comprising a mixture of differentiable cells and desired cell lineage can be produced.

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

The invention also relates to a kit, the kit comprising at least one compound capable of stimulating ErbB2-derived tyrosine kinase activity and a basic 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, variant or functional fragment 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, variants or functional fragments thereof. In another embodiment, the kit includes serum albumin.
In yet another embodiment, the kit comprises serum, at least one insoluble substrate described herein, and / or at least one disaggregation solution.

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

  Throughout this application, various documents are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety in order to more fully describe the state of the art in the technical field of the present invention.

Examples The human embryonic stem cell line BG01v (BresaGen, Inc., Athens, GA) was used in some of the experiments described herein. The BG01v hESC line is a karyotypic mutant cell line. The cell line shows a stable karyotype containing specific three chromosomes (karyotype: 49, XXY, +12, +17). Parent cultures were maintained as described previously (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 MATRIGEL® or fibronectin coated dishes in DMEM: F12 and 20% KSR, 8 ng / mL FGF2, 2 mM L-glutamine, 1 × non-essential amino acids, 0.5 U / mL. Collagenase in conditioned medium (Sigma, St. Louis, MO, USA) from mouse embryonic fibroblasts (MEFs) (MEF-CM) containing penicillin, 0.5 U / mL streptomycin, 0.1 mM β-mercaptoethanol Grown in passage.

  The defined culture (DC) medium tested here is 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 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 factors were used. The factors include FGF2 (Sigma), LongR3-IGF1 (JRH Biosciences), Heregulin-β EGF domain (HRGβ, Peprotech), TGFβ (R & D systems), 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 a culture as defined above 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 containing 10 ng / mL HRG-β, 10 ng / mL activin A, and 200 ng / mL LR-IGF1. The DC-HAIF, DC-HAI, and LR-IGF1 components are of course replaced with IFG1.

  MATRIGEL®-coated dishes were grown about 1% in cold DMEM / F-12 from Growth Factor Reduced BD MATRIGEL® Matrix (BD Biosciences, Franklin Lakes, NJ, USA). Prepared by diluting to a final concentration range of 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 for 1-2 hours at room temperature or at least overnight at 4 ° C. Cultures were stored at 4 ° C for up to 1 week. The MATRIGEL® solution was removed immediately prior to use. For the conditions tested, parent cultures were plated in 6 well dishes for comparison of multiple conditions. Cultures were typically plated at direct test conditions. Cultures were evaluated daily starting 4-5 days after plating and graded based on morphological criteria. A rating scale of 1 to 5 examines the whole culture and calculates the overall proportion of undifferentiated colonies, their relative size, the proportion of colonies showing obvious differentiation or the portion of the cornie I did. Grade 5 represents an “ideal” culture with large undifferentiated colonies and negligible differentiation. Grade 4 shows a very good culture, but some obvious differentiation. Grade 3 shows an acceptable culture, but approximately half of the colonies show obvious differentiation. Grade 2 is mainly differentiated and there are sometimes 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 cultures for longer periods under these conditions.

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

  Real-time RT PCR was used to show the expression of ErbBl-3, neuregulin, and ADAM-19 in BG01v cells (FIG. 1). BG01v cells cultured in DC medium as described above, including 100 ng / mL LongR3-IGFl (LR-IGFl), 8 ng / mL FGF2, 1 ng / mL activin A are harvested and RNA is the manufacturer's instructions According to 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 method and Taqman method for Taqman (Tsman) and ADCT19 (Hs00224960_ml), EGFR (Hs00193306_ml), ErbB2 (Hs00170433_ml), ErbB3 (Hs00176538_ml), NRGl (Hs00247620_ml), OCT4 (Hs00742896_sl) GAPDH was used in TaqMan universal PCR (Applied Biosystems). A real-time amplification plot showing the expression of these transcripts in undifferentiated BG01v cells is shown in FIG.

Example 2-Inhibition of ErbB2 slows BG01v cell proliferation

  The EGF domain family of ligands binds to the ErbB family of receptor tyrosine kinases. To examine the known inhibitory effect of ErbB tyrosine kinase in hESCs, BG01v cells were grown in a 1000 well diluted MATRIGEL® 6 well tray in 100 ng / mL LongR3-IGF1, 8 ng / mL FGF2 , And 1 ng / mL activin A in defined media (DC). The next day, DMSO (carrier control), 50 nM-20 microM AG1478 (ErbBl inhibitor), or 100 nM-20 microM AG879 (ErbB2 inhibitor) was added along with fresh medium. Cells were cultured for 5 days with daily media changes. Cultures were fixed and stained for alkaline phosphatase activity.

  Subconfluent colonies of AP + BG01v cells were observed in control and AG1478 cultured cells (FIGS. 2A and 2B). Neither DMSO nor AGl478 (50 nM-20 microM) was shown to have a clear effect on cell proliferation. AG879, however, substantially inhibited 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 defined medium containing heregulin

  ErbB2 and ErbB3 expression and growth inhibition at AG879 suggested that BG01v cells are active for endogenous ErbB signaling and that they can respond to exogenous HRG-β. BG01v cells grow on 1: 1000 diluted MATRIGEL® in DC media containing 10 ng / mL HRG-β, 200 ng / mL LongR3-IGF1, 8 ng / mL FGF2, and 10 ng / mL activin A (FIGS. 3A and B). Cells were grown for more than 4 generations 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-IGF1, and 10 ng / mL activin A for 2 or more days. These cultures grew normally and showed very low idiopathic differentiation. This indicates that BG01v cells could be maintained under defined conditions in the absence of FGF2 and in the presence of HRGβ.

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

  RT-PCR showed that mESCs expressed ADAM19, neuregulin 1 (Nrgl), and ErbB1-4 (FIG. 4A). In mESCs, ErbB2 and 3 were shown to be expressed at higher levels than ErbB1, and low levels of ErbB4 were detected. These data suggest that endogenous HRG-β could be involved in promoting mESC self-renewal.

  The expression of ErbB receptor transcripts in mouse fetal fibroblasts (MEFs) was also examined (FIG. 4B). MEFs are a heterogeneous population of cells obtained from E12.5-13.5 viscera that have historically been used to maintain mouse and human EC cells and embryonic stem cells. The expression of Nrgl and Adam19 in this population suggests that the HRG-β ectodomain is also present in MEF conditioned media and can have 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 standard in 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. Maintained with βME and passaged with 0.5% trypsin / EDTA. 2 × 10 ⁵ cells / well were plated in 6-well trays on MATRIGEL® diluted 1: 1000. The day after plating, DMSO (Carrier Control), 1-50 microM AG1478, or 1-50 microM AG879 was added along with fresh media. The cells were cultured for an additional 8 days with daily media changes. The culture was then stained for immobilized alkaline phosphatase activity.

  DMSO and 1-50 microM AG1478 had no obvious effect on cell proliferation. “Sub-confluent” colonies of alkaline phosphatase positive mESCs were observed (FIGS. 5A-C). However, AG879 could substantially inhibit cell growth at 50 microM (compare FIGS. 5D and 5F) and slow growth at 20 microM (FIG. 5E). MESCs grown on AG879 did not show differentiation and maintained pluripotency morphology and alkaline phosphatase activity.

  The results indicated 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 times higher for mESCs than that for BG01v cells grown in defined conditions. Whether serum used in mESC conditions hindered 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 Shows what you can do.

  Another highly selective inhibitor of ErbB2 tyrosine kinase, tyrphostin AG825 (Murillo, et al. 2001 Cancer Res 61, 7408-7412) was used to investigate the role of ErbB2 in human ESCs. AG825 significantly inhibited the proliferation 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 longer than 5 days (not shown). Western blot shows that AG825 inhibited autophosphorylation of ErbB2 at tyrosine-1248 in starved / heregulin (HRG) pulsed hESCs growing in DC-HAIF-starved / heregulin (HRG) pulsed hESCs. (FIG. 6B). Thus, ErbB2 division signaling severely suppressed hESC proliferation. To establish hESCs at defined growth conditions, cultures were directly passaged from CM conditions to DC-HAIF and showed minimal idiopathic differentiation (FIG. 6C). Colony and cytometric assay methods confirmed that LongR3-IGF1 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 pulsed starvation medium (FIG. 6F). .

Example 5, Mouse ES cell culture under defined conditions

To further investigate the role of HRG / ErbB2 signaling in mouse embryonic stem cells, the growth of Rl ES cells was examined in DC medium using a combination of growth factors. 1 × 10 ⁵ cells / well are plated into 6-well trays and coated with 0.2% gelatin, 10 ng / mL HRG-β, 100 ng / mL LongR3-IGF1, 1 ng / mL Activin A, 1000 U Incubated in DC containing a combination of / mL mouse LIF or 10 ng / mL 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 defined medium without growth factors. See Table 1.

Quantitative measurements were performed on selected combinations of 1: 1000 MATRIGEL® in 6 well trays and 10 or 50 ng / mL HRG-beta, EGF, 1000 U / mL LIF or 10 ng / mL BMP4. X10 ⁵ cells / well were plated. The culture was grown and fixed for 8 days. And the number of alkaline phosphatase colonies was counted (FIG. 7A). No colonies were observed at the defined conditions and without growth factors. And fewer than 45 colonies were observed with the combination of HRG-β, HRG-β / EGF, and HRG-β / BMP. 1358 colonies were observed with LIF alone, 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 indicated that under defined conditions, only LIF provided a substantial pluripotency signal. And HRG-β showed a large synergistic effect with LIF, and in this analytical evaluation, the number of proliferating mESC colonies was more than doubled. Moreover, the low magnification image of this analytical evaluation shows this synergistic growth promoting effect (FIGS. 7B-G).

Example 6 Characterization of pluripotency of human embryonic stem cells (hESCs) maintained in DC-HAIF Several approaches were used to confirm the maintenance of the formation of hESCs in DC-HAIF. BG02 cells cultured in 6-month (25th) DC-HAIF maintained the possibility of forming complex teratomas (FIG. 8A) and were representative of the three germ layers in vitro. 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 transcripts were detected in BG02 cells from passage 10 to 32 grown in DC-HAIF and in BG02 cells grown in passage 64 in CM. There were generally about 10364 transcripts in all populations including known hESC markers such as CD9, DNMT3, NANOG, OCT4, TERT and UTF1 (not shown) (FIG. 8C). A high correlation coefficient was observed in CM and DC-HAIF culture comparisons (R ² select = 0.828) and in early and late passage cells (R ² select = 0.959) (FIG. 8D). Hierarchical cluster analysis showed that BG02 cells maintained in DC-HAIF were firmly retained and had 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. Thus, cells maintained with the compositions of the invention can maintain pluripotent key markers. Therefore, the composition of the present invention can be used as a simple medium for supporting self-renewal of differentiable cells.

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

  To investigate the role of ErbB2 signaling and develop defined media for hESCs, DC-HAIF cultures were grown with growth factor-reduced 1:30 MATRIGEL®-coated culture dishes Initially spread over. It could be successfully maintained in the long term on a substrate diluted 1: 200 or 1: 1000 (FIG. 9A). Alternatively, 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. BG02 and CyT49 hESCs could be maintained for longer than 5 passages.

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

  Several research groups have shown that certain triploidy, notably hChrl2 and 17, accumulate in hESCs under some optimal culture conditions (Baker et al., 2007 Nat. Biotech. 25 (2): 207-215). The triploid appearance appears to be most directly related to inadequate cell survival when the culture is separated into single cells at passage, and these aneuploid cell harboring Strong selective growth benefits estimated for are provided to the cells. Conversely, hESCs grown on DC-HAIF in one embodiment of the present invention maintained high viability upon plating after being separated into single cells (FIGS. 10A-D). BG01 and BG02 cells were maintained in normal karyotype after passage> 18 and 19 respectively under the ACCUTASE® trademark (FIG. 10E). Maintenance of normal karyotype in the cells indicated that disaggregation of hESC cultures into single cells did not lead to these trisomic accumulation of hESCs originally maintained with DC-HAIF. BG01 and BG02 cultures are also passaged by disaggregation into single cells using multiple passage reagents (FIG. 11). The medium was split and karyotyped for 5 passages with ACCUTASE®, 0.25% Trypsin / EDTA, TrypLE, or VERSENE®. The data show that culturing and passaging of hESCs with the compositions of the present invention maintained a normal karyotype in at least two human embryonic cell lines using various cell dissociation reagents.

Large scale expansion of undifferentiated hESCs is also possible using the composition of the present invention. The first confluent culture of BG02 cells in a 60 mm plate was grown in 4 passages to more than 1.12 × 10 ¹⁰ cells in one experiment in DC-HAIF for 20 days. It was. The culture remains undifferentiated and according to flow cytometry, more than 85% of the cells in the batch are expressing multipotent markers such as OCT4, CD9, SSEA-4, TRA-1-81 Was maintained (FIG. 12A). In addition, expression of other markers of pluripotency was also observed by RT PCR analysis. On the other hand, markers of differentiated lineage α-fetoprotein, MSX1, and HAND1 were not detected (FIG. 12B). Fluorescence in situ hybridization analysis showed that cells cultured and passaged in DC-HAIF were hChrl2 (98% 2-copy), hChrl7 (98%, 2-copy), hChrX (95%, 1-copy). The expected copy number was maintained for hChrY (98%, 1-copy) (FIG. 12C). Karyotype analysis also showed that normal ploidy chromosome content and banding profiles 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 a physiological excess of insulin that can stimulate both IR and IGFIR. In order to distinguish the activity exerted by insulin and insulin substitutes compared to IGFl, hESCs are cultured in defined media conditions of physiological concentrations of these growth factors. Insulin and IGFl concentrations are from about 0.2 to about 200 ng / mL and cell proliferation is monitored by counting cells after 5 days. Successful spreading media are passaged 5 times in succession. While physiological concentrations of insulin do not support expansion of hESC cultures, physiological concentrations of IGFl support expansion of hESC cultures. This indicates that the activity of insulin or insulin surrogate cannot replace IGFl, and IGFl and insulin (or insulin surrogate) exert separate classes of biological activity with respect to action on hESCs.

Example 10-Method for screening the effect of supplements

To initially assess the effects of vitamins B ₁₂ and B _{6 on} the growth or differentiation of hESCs growing at intermediate density, BG02 cells were used at 1 × 10 ⁵ cells / well using ACCUTASE®. , Plated with defined culture (DC) medium in 6 well trays and split. DC medium contains 10 ng / mL HRG-β, 200 ng / mL LongR3-IGF1, and 10 ng / mL FGF10. Vitamin B6 (0.5 microM) and / or vitamin B ₁₂ (0.5 microM) is added to the test well. The number of cells in each condition is counted after 7 days. The number of cells and colonies in both experimental and control wells gives a prospect of the effect on vitamin B ₁₂ and B ₆ cell growth.

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

Example 11-Medium of hESCs in the absence of FGF2

  BG02 cells were maintained in DC-HAI for a long period (passage 20) (FIG. 13A). BG01 cells were also passaged continuously in DC-HAI. Both were in the absence of FGF2. The culture did not deteriorate or showed obvious differentiation and showed normal expansion of undifferentiated colonies throughout the culture period. Maintenance of normal male karyotype in BG02 culture was shown after 6 passages in DC-HAI (FIG. 13B, 20/20 metaphase 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 experimental protocol. Sample amplification was performed using an Illumina RNA Amplification kit with 100 ng of total RNA. Labeling was then achieved by adding biotin-16-UTP (Perkin Elmer Life and Analytical Sciences) in a 1: 1 ratio with unlabeled UTP. Labeled amplifications (700 ng per array) were prepared according to the manufacturer's instructions (Illumina, Inc.) with Illumina Sentrix Human-6 Expression Beadchips containing 47,296 transcription probes. ). The array was scanned with an Illumina Bead Array Reader confocal scanner, and primary data processing, background removal, and data analysis were performed using the Illumina BeadStudio software at the manufacturer's instructions. 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 previously described (Liu et al., 2005, BMC Dev Biol 6:20), and a similarity metric using differentially expressed genes identified by ANOVA. As based on average concatenation and Euclidean distance (p <0.05). Details of the sensitivity and quality control tests used in array manufacturing and a detailed description of the algorithms used in the bead studio software are available from Illumina Inc (San Diego, Calif.). Most of the detected transcripts were expressed in both DC-HAIF and DC-HAI BG02 cultures and contained known hESC markers such as CD9, DNMT3, NANOG, OCT4, TERT and UTF1 (not shown). A high correlation coefficient was observed in a comparison of DC-HAIF and DC-HAI cultures (R ² select = 0.961) (FIG. 14). Hierarchical clustering analysis shows that BG02 cells maintained in DC-HAI are tightly grouped and maintained in DC-HAIF as well as multiple culture formats of BG02 and other hESC lines. Possessed similar similarity to the cells (FIG. 15). These data are consistent with previous analysis showing that undifferentiated hESCs are clustered tightly compared to embryoid bodies or fibroblasts. (Liu et al., 2006, BMC Dev Biol 6:20)

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

  To investigate whether exogenous FGF2 was required for single cell passage, BG01 cells were passaged with ACCUTASE® and only 10 ng / mL HRG-β and 200 ng / mL LongR3-IGF1 (DC-HI) Grown under defined conditions including. These DC-HI cultures were maintained for 10 passages and showed no apparent differentiation or slowing of proliferation.

  These studies clearly showed that exogenous FGF2 is not required when maintained in defined media containing hESCs minimal containing heregulin and IGFl. Furthermore, the culture medium without FGF2 possessed the basic properties of pluripotency including in vivo differentiation into mesoderm, endoderm and ectoderm and transcriptional profile.

Example 12-Suspension culture

The starting medium of BG02 cells is maintained in DC-HAIF medium on a dish coated with 1: 200 matrigel as described herein, and passaged and divided with ACCUTASE®. It was. To initiate suspension culture, BG02 cells were dissociated with ACCUTASE®, and 1.6, 3, or 6 × 10 ⁵ cells / mL (0.5 mL in 3 mL volume) in DC-HAIF medium. 1 or 2 × 10 ⁶ cells) in low attachment 6 well trays. Trays in an incubator with humidified 5% CO _2, was placed on a rotating platform at 80-100Rpm. Under these conditions, hESCs fused morphologically to viable globules within 24 hours.

  On the second day and every day thereafter, the medium in the wells was changed. The suspension population continued to grow and grew with time without obvious signs of differentiation (Figure 17). Some of the spheres remained aggregates over the course of the culture, but some populations were much larger than most. Furthermore, non-spherical aggregates could be observed during the coalescence process during the first few days of culture. Limited to this continuous population, 38 microg / mL DNase I was included in the first 24 hour suspension culture. However, this approach appeared to be useful for a relatively large initial assembly, as a smaller population was formed in the presence of DNaseI. However, it is not clear whether DNase I treatment reduces the sphere coalescence that follows, and exposure to DNase I consistently makes these populations stiffer and destroyed upon separation.

Suspension cultures were disaggregated approximately every 7 days with ACCUTASE® to create new spheres. Density differed by experiments, between the density in the range (1.6-6 × l0 ⁵ cells / ml) at-made balls is longer than 12 passages between or longer than 80 days, morphology of differentiated Could be maintained in the culture medium without any signs. FISH analysis of serially passaged suspended hESCs was performed to calculate 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 differentiable cells in suspension culture

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

Cell count was used to assess the relative growth rate of cells in suspension culture compared to adherent culture. In this experiment, adherent cultures of BG02 cells were passaged with ACCUTASE® and approximately 1 × 10 ⁶ cells were placed in suspension or adherent culture wells. Individual wells were counted for 1-6 days and plotted on a log scale (Figure 18). In adherent cultures, a higher initial percentage of hESCs after 24 hours (about 90% vs. about 14%) was shown, but the growth rate was thereafter comparable. This indicated that hESCs can grow almost as rapidly in traditional adherent and suspension cultures. Cell counts performed during the passage allow to measure the amount of expansion possible with this simple suspension system. In some cultures, seeding of 5 × 10 ⁵ cells generated more than about 10 ⁷ cells after 7 days. The expansion after 7 days in suspension culture was equivalent to an expansion of about 20 times or more, and the maximum expansion observed was about 24 times.

Example 14-Properties of differentiable cells expanded in suspension culture

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

Example 15-Addition of apoptosis inhibitor in suspension culture

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

A suspension population of BG02 cells was formed by seeding 2 × 10 ⁶ single cells in 6-well dishes in 3 mL DC-HAIF medium and placing them on a 100 rpm rotating platform in an incubator (Table 2, Experiment A). ). 10 μM Y27632 ROCK inhibitor was added to the test wells according to the experimental design, observed daily and counted after 24 hours (Day 1) and after 4 or 5 days. As shown in FIG. 20, the addition of Y27632 had a profound effect on the initial assembly phase of suspension culture. A much larger population was formed in the presence of Y27632 compared to cells assembled in medium without inhibitor (FIG. 20). Cell counts confirmed that more viable cells were present when the inhibitor was present (Table 2, Experiment A). This difference in cell number persisted over the course of the culture period. Also, more cells were observed on day 4 compared to cultures without inhibitor. Similar to previous suspension culture experiments, cells exposed to Y27632 could be passaged continuously and maintained in an undifferentiated state (not shown). When splitting again, almost twice as many cells were observed as those treated with Y27632 (Experiment A in Table 2). RT-PCR analysis showed that BG02 cells grown in suspension culture in the presence of Y27632 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 adherent cultures were similar after the initial 24 hours (Table 1). 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 The viability count was not negatively affected. Survival of 1.4 × 10 ⁷ (+ Y27632) and 1.8 × 10 ⁷ (+/− Y27632) when inhibitors were present compared to 3.9 × 10 ⁶ cells in untreated cultures Cells developed. This analysis confirmed that Y27632 had the greatest impact during the first 24 hours of suspension hESC culture.

Experiments were performed to see if it was possible to reduce the number of cells used to seed suspension cultures due to the improved survival and aggregation observed in the presence of Y27632. Experiment C). Previous experiments have shown that seeding of embryonic stem cells at low density of approximately 5 × 10 ⁵ cells or less per 3 mL DC-HAIF does not work well. To confirm whether the addition of ROCK inhibitor allowed cell seeding at low density, a range of cell concentrations (2 × 10 ⁶ cells to about 1 × 10 ⁵ cells) was achieved with 3 mL DC-HAIF. Used to seed suspension cultures with 6-well tray cells. 10 microM Y27632 was added under all conditions and cell number and viability were assessed on day 5. Successful assembly and expansion were observed even at low seeding densities. Even with media that was only seeded with 1 × 10 ⁵ cells, approximately 13-fold expansion of viable cells was observed. Thus, inhibition of ROCK by Y27632 facilitated early survival of hESCs at a lower density in this suspension culture system.

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

Example 16-Suspension culture in various media To determine whether suspension culture of embryonic stem cells is possible in the absence of FGF2 and / or Activin A, the presence of these elements varies the number of embryonic stem cells In different medium. Table 3 shows the cell count results of the suspension culture, which was also successfully performed in the absence of exogenous FGF2 (HAI condition) or in the absence of exogenous FGF2 or activin A (HI condition). Indicates that expansion was possible. The addition of Y27632 increased cell yield in 5 days under all conditions. Furthermore, the cells in each medium did not show morphological signs of differentiation and were successfully passaged.

Example 17-Optimized Shear Rate at Increased Survival, Uniform Density, and Size of Suspension Cell Aggregation According to the systems, methods, and devices disclosed herein, It is contemplated that all cell lines that can be maintained will benefit and be available. Cells include, but are not limited to, human cell lines CyT49, CyT203, Cyt2S, BG01, BG02, mammalian cells including mice, dogs, non-human primate stem cell lines, and others.

  The results provided herein show that cell growth and differentiation can be maintained or attenuated at a controlled level, depending on the operating parameters associated with the reactor, particularly the culture flow rate and shear force. Show. The shear force applied to the cell culture can have a significant effect on cell proliferation. A symmetric system such as a rotating platform used herein provides uniform, primarily laminar shear stress around the vessel, and an asymmetric system such as a stirred tank bioreactor and its mounting is locally It has a region of turbulent flow characterized by high-speed shear stress. If the bioreactor or cell culture device is not a symmetric system, the direction of culture flow affects both the nature and extent of shear stress resulting from rotation.

Of course, the optimal number of rotations depends on the specific medium, the number of cells in the medium, the viscosity of the culture, the type of medium, the robustness of certain cells in suspension (some cells are larger than others) It can withstand shearing force). The optimum rotational speed can be easily determined according to a specific set of conditions. The number of revolutions described and assumed herein is 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 the cells grew and differentiated were attenuated.
The following is a general method that can successfully maintain hES cell aggregate cultures or differentiated hES cell aggregate cultures. One skilled in the art can optimize the size and shape of the cell aggregate based on the description provided herein.

Table 4 below shows the shear rate and stress associated with the cell aggregate diameter (μm).
Human embryonic stem cells were assembled for 1, 2, 3, and / or 4 days at various rotational speeds using an orbital rotator (Bamstead 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 rate experienced by the cell aggregates depending on the cell aggregate diameter.

To determine how the rotational speed controls the diameter of the ES assembly, the ES assembly was generated by spinning at 100 rpm, 120 rpm or 140 rpm. Aggregate diameter was quantified from 5 × phase contrast images obtained from rotating cultures after 2 days. In the 100 rpm culture, the average diameter +/− SD was 198 μm +/− 21 μm. In the culture at 120 rpm, the average diameter +/− SD was 225 μm +/− 28 μm. In the culture at 140 rpm, the average diameter +/− SD was 85 μm +/− 15 μm. Each diameter distribution is statistically significant using ANOVA and Tukey Multiple Comparison posttest (p <0.001). 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 assembly, the shear rate is approximately 30 seconds ⁻¹ , whereas at 140 rpm it is approximately 80 seconds ⁻¹ , a threefold increase. Typically, rotational speeds above 140 rpm were greater, resulting in less uniform hES cell aggregates. Cell aggregate cultures are initially cultured at a reduced rotational speed, eg, about 60 to 80 rpm for about 1 day, and then at higher rotational speeds (eg, 100 rpm-140 rpm or higher) Can be cultured without adversely affecting size and shape.

  It should be noted that although the cell aggregate diameter varies with shear rate, there is no significant effect on gene expression under various conditions, ie different rotational speeds and / or different sizes and shapes of cell aggregates. is important. That is, the signature markers observed for pluripotent hES cells or hES cell-derived cell types (eg, final endoderm, foregut endoderm, PDX1 endoderm, pancreatic endoderm, and endocrine cells) Consistent with that described in the d'Amour et al application and related applications referenced and incorporated herein above.

In order to determine the effect of rotational speed, shear rate, and shear stress on cell survival or cell viability, it was shown that survival was improved by one day at a reduced rate (eg, 60 rpm to 80 rpm). For example, cell survival ranged at least 60% or more at a rotational speed 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 higher rotational speeds (eg, 100 rpm or higher). There was also significant division and disaggregation when cell aggregates were cultured at higher rotational speeds (eg, 140 rpm or higher). At the same time, however, these data indicate that cell survival is increased when cell aggregates are initially cultured at a reduced rotational speed for at least one day, and when the rotational speed is increased from 100 rpm to 140 rpm, It indicates that there was no significant reduction in survival.
However, differentiation at a rotational speed of less than 140 rpm is preferred.

  The culture volume also affects the shear rate and shear stress, as discussed above, which affects the size and shape uniformity of the cell aggregates. For example, when single cell suspension culture is started and a cell aggregate is formed with 6 mL, a cell aggregate with a more uniform size and shape can be obtained as compared with that started with 4 mL. Refer to FIG. When cultured at 4 mL, the diameter of cell aggregates varies from less than 50 microns to 250 microns or more, but when cultured at 6 mL, the diameter becomes a narrower range, and the diameter of cell aggregates ranges from 50 microns to 200 microns. It became below micron. The described cell aggregates 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 the medium is almost independent of the stage at which cell assembly suspension culture is initiated.

  Furthermore, hES cell aggregates can be cultured under a variety of different medium conditions. For example, StemPro®-containing medium, DMEM / F12-containing medium; 20% Knockout serum replacement-containing (KSR, Invitrogen) DMEM / F12-containing medium; 20 ng / mL FGF (R & D Systems) and 20 ng / mL Activin A ( HES cell assembly cultures can be maintained in StemPro® and DMEM / F12 medium further comprising R & D Systems; StemPro® and DMEM / F12 medium further comprising 10 ng / mL Heregulin B; Alternatively, any of the culture media referred to in the present specification and a commercially available culture media can be used in a state supplemented with non-foreign KSR (Invitrogen). Finally, cell aggregates were cultured and produced in the above medium and also without exogenous FGF.

Example 18-hES cell aggregates in suspension culture can differentiate into endoderm lineage type cells d'Amour et al. (2006) and US patent application numbers 2005/0266554, 2005/0158853, 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), foregut endoderm, and PDXl endoderm To differentiate. The above references are referenced and incorporated in their entirety as part of this specification.

  Briefly, undifferentiated pluripotent hES adherent (plate) cells can be obtained on mouse embryonic fibroblast feeder layers (Millipore, former Chemicon or Specialty Media) or 20% KnockOut serum replacement (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 top of a 60 mm plate (0.1-20% final concentration; Valley Biomedical) coated with human serum in DMEM / F12 (Mediatech) supplemented with mL to 20 ng / mL recombinant human FGF2 (R & D Systems) Maintained at. Alternatively, the medium can be supplemented with xeno-free KSR (Gibco) and human serum. In addition, human serum was added to the culture after 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. Day 0 (d0) adherent pluripotent hES cells express a high level of pluripotency protein marker, OCT4. See FIG. 1, panel A, plate control in d0.

Cells were re-passaged manually or enzymatically, substantially as described in d'Amour et al., Supra. The suspension culture was separated and transferred to a conical tube and centrifuged at 1000 rpm for about 5 minutes. The supernatant was removed and a standard cell count was performed using a BiCell Cell Analyzer. The typical number of cells from a 60 mm plate varies from 3 × 10 ⁶ to 12 × 10 ⁶ and depends on the number of days and cell lines in the medium prior to passage. Once the number of cells in the primary cell suspension is determined, the suspension is further diluted as described above with medium containing StemPro® or non-foreign KSR, and 1 × 10 ⁶ cells / mL. Diluted to a final volume of. This volume can be increased to> 4 × 10 ⁶ cells / mL but requires more frequent feeding. The ROCK inhibitor Y27632 (Axxora) was added to the cell suspension to a final concentration of about 1-15 microM, typically 10 microM. The tube was then mixed by gentle inversion. In some cases, Y27632 was not added to the suspension to control the rate of aggregate formation. The resuspended cells are then equally distributed into each well of a low binding 6-well dish (approximately 5 mL of cell suspension per well) at about 100-140 rpm for about 1-4 days prior to differentiation. Placed on a rotating platform.

  During this culture, hES cell aggregates are formed and are added to the culture by replacing 4 mL of medium with 4 mL of Y27632 minus Y27632 from fresh StemPro® medium at least 1-2 times daily. Feeding daily or supplemented with non-foreign KSR in any of the media described above. Media exchange ("feeding") should be performed as fast as possible to avoid disruption of the aggregate and break the surface tension that can float the aggregate during rotation. Also, cell aggregates should not be removed from a rotating platform or device for extended periods of time 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 differentiated directly as aggregates in suspension, as described in D'Amour et al. (2006) above. Briefly, StemPro® (minus Y27632) medium or any of the above medium supplemented with non-foreign KSR is removed from the well (eg, aspirated) and 5 mL of hES cell aggregates Is 5 mL of serum-free RMPI (Cat. 15-040-CV; Mediated.), Penicillin / streptomycin (Invitrogen), and glutamax (also RMPI, Pen / Strep, and glutamax medium) Washed) with 0% FBS, 1% pen / strep, 1% glutamax. And 6-well dishes were put back on the rotating platform 1-2 minutes before removing the washing medium. This was repeated at least twice or until insulin and / or IGF-1 was sufficiently removed. Necessary for maintaining pluripotency and ES self-renewal, because the same elements are detrimental to controlled, synchronized, lineage-derived differentiation.
With the addition and removal of various exogenous mitogens, differentiation into all endoderm lineage was performed substantially at 100 rpm as described in d'Amour et al. (2006). Further details are described below.

Differentiation into final endoderm (stage I) Human ES cell aggregates were first day RPMI, 100 ng / mL activin A and variable concentrations of FBS (US Defined FBS, HyClone, Catalog No. SH300700.03), and 25 ng / mL-75 ng / mL Wnt3a, Day 2 and Day 3 (d0 to d2) are further differentiated in RMPI, 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 experimental design is used or desired, most differentiation experimental FBS concentrations are 0% for the first 24 hours (d1) and 0. 2 for the second 24 hours (d2). For 2% (d2) and the third 24 hours (d3), it is 0.2% (d3). Preferably, a two day stage 1 experimental design is performed.

  QPCR analysis of hES-derived cell aggregates in suspension culture at the end of the two-day stage 1 experimental design is more efficient than the adherent plate control towards the final endoderm of the hES aggregates. 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 endodermal cells and the adherent hES cell culture were used as controls. Increased expression levels of SOX17 and FOXA2 were observed in suspension and adherent cell aggregates compared to undifferentiated hES cell aggregates and adherent plate controls. See FIG. 22, Stage 1 (d2) of Panel C (SOXl7) and D (FOXA2). Furthermore, the expression levels of genes associated with contaminating SOX7, extraembryonic and organ endoderm were significantly reduced in the final endoderm cell population as compared to the final adherent plate control of the endoderm. See FIG. 22, panel 1 stage 1 (d2).

  Flow cytometric analysis using CXCR4 and HNF3beta (FoxA2) proteins showed 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 core. -Shown to yield a positive population.

  To further evaluate the efficiency of hES cell assembly differentiation, cryosections of ES-derived cell assemblies were examined for immunosociation and confocal microscopy for SOXl7 and HNF3beta expression. Image analysis of stained frozen sections showed that over 90% of all cells at the end of stage 1 (final endoderm cells) expressed HNF3beta and / or SOX17.

  All of these data were able to achieve highly efficient differentiation of embryonic stem cells as cell aggregates and based on the expression level of the final signature definitive endoderm marker in the identification region, Compared to differentiation, as described herein, the method for producing the final endoderm is shown to be more efficient.

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

  Human definitive endoderm cell aggregates from stage 1 are easily washed with PBS + / + and then further 2% FBS, 25 ng-50 ng / min with RMPI, pen / strep and glutamax media. Differentiation was further performed for 2 to 3 days among those containing mL KGF (R & D Systems). In some experiments, 5 microM SB431542 (Sigma Aldrich Inc.) or 2.5 microM 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 HNF1β and HNF4alpha were observed in cell aggregate cultures compared to adherent plating controls. See FIG. 22, panel E (HNF1B) and stage 2 (d5) of panel O (HNF4alpha). The method of producing specific stage 0, 1, 2, and 5 hES or hES-derived cell aggregates (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 corresponding stage of the adherent plate control culture from which they are derived. For example, in stage 1, a differentiated cell aggregate (“dAggs”) suspension culture is initiated from an adhesive plate in stage 0 and the medium described herein for about 24 hours on a rotating platform from 100 rpm to 140 rpm. Incubate with either. These differentiated cell aggregates were further differentiated into stage 1 final endodermal cells with corresponding adherent plate controls. FIG. 22, panel O shows that there is no significant HNF4alpha (HNF4A) expression in either one stage differentiated cell population or the adherent plate control. In contrast, a similar method was performed for stage 2 samples to produce HNF4A at increased levels of expression. In the stage 5 sample, the expression of HNF4A is also solid.

  Furthermore, compared to plate culture controls, the expression level of genes associated with extraembryonic endoderm (SOX7) was significantly reduced in hES-derived cell aggregate cultures. See FIG. 22, Panel L, Stage 2 (d5). The results show that directed differentiation of PDX1-negative foregut endoderm cells by cell aggregates in suspension culture removes extraembryonic endoderm contaminants.

  All of these data indicate that directed differentiation of hES cell aggregates is highly efficient and produce foregut endoderm cells based on the expression level of PDX1-negative foregut endoderm markers in the discriminating region The method to do shows improved compared to differentiation in adherent plate cultures.

Differentiation into PDX1-positive foregut endoderm cells (stage 3)

  Foregut endoderm cells from stage 2 are serum-free RMPI for 1-3 days, glutamax (Invitrogen), penicillin / streptomycin (Invitrogen), 0.5X B27-supplement (Invitrogen / Gibco), 1 microM to 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 KAAD-cyclopamine, and 50 ng / mL Differentiated by adding Noggin (R & D systems). Alternatively, 0.2 microM to 0.5 microM RA and 0.25 nM KAAD-cyclopamine were added to the medium for 1 day. Furthermore, in some experiments, RA or KAAD-cyclopamine was not added to the cell assembly culture. In other embodiments, an effective concentration of BSA of 0.1-0.2% was added.

  Increased expression levels of PDXl were observed in hES-derived cell aggregates compared to adherent plating controls. See FIG. 22, panel F (PDXl), stage 3 (d8). Furthermore, compared to plate culture controls, the genes associated with extraembryonic endoderm (SOX7) and the expression level of organ endoderm (AFP) were significantly reduced in hES-derived cell aggregate cultures. See FIG. 22, panel L (SOX7), panel N (AFP), stage 3 (d8). Thus, directed differentiation producing PDXl-positive foregut endoderm cells by cell aggregates in suspension culture has been shown to remove extraembryonic endoderm contaminants.

  These data indicate that directed differentiation of hES cell aggregates is highly efficient, and based on the expression level of PDX1-positive foregut endoderm markers in the identification region, PDXI-positive endoderm cells The method for manufacturing is shown to be improved compared to differentiation in control adherent plate cultures.

Differentiation into pancreatic endoderm or pancreatic endocrine precursor 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 for 4-8 days with DMEM + 1XB27 supplement alone or in combination with any or all of the following elements: Noggin (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, noggin 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 adherent plating controls corresponding to ES cell-derived aggregates. See FIG. 22, Panel F (PDXl), Panel G (NKX6.1), and Panel P (PTFA1), Stage 4 (d11). In FIG. 22, panel P, to determine whether hES and / or hES-derived aggregates in suspension culture are affected by the number of cells in the adherent plate culture from which they are derived. A bar graph represents the results from the method. Only panel P shows results for 1 × 10 ⁷ cells, but cell aggregate suspension cultures were started with various seed counts (eg, 1 × 10 ⁶ to 2 × 10 ⁷ cells). All were substantially similar and produced cell population cultures with good viability and almost no cell death. For example, in stage 4, differentiated cell aggregate suspension culture cells (“dAggs”) are started with d5 (stage 2) adherent plate cultures and again any of the media described herein. Were incubated on a rotating platform at 100 to 140 rpm for 24 hours. These differentiated cell aggregates were further differentiated into stage 4 pancreatic endoderm type cells expressing PTFlA (panel P). There is an increased expression of PTFlA compared to the corresponding stage 4 adherent plate control.

  Furthermore, compared to adherent plating controls, the expression level of AFP was significantly reduced in hES-derived cell aggregates. See FIG. 22, panel N, stage 4 (d11). Thus, directed differentiation that produces 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 hES-derived cell aggregates had at least 53% CHG-positive, at least 40% NKX6.1 and CHG. It was shown to give cell aggregates that were co-positive and small amounts of HNF3beta and other types of cells.

  A cryosection of the hES-derived population was examined at the end of stage 4 using immunocytochemistry and confocal microscopy for expression of NKX6.1, PDXl and NKX2.2. Image analysis showed highly efficient differentiation of aggregate cells into pancreatic endoderm (or PDX1-positive pancreatic endoderm). Almost all cells expressed PDXl and a large population of cells expressed NKX6.1 (about 40% cells) and / or NKX2.2 (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, medium was supplemented with human serum (Valley Biomedical) or fetal bovine serum in the range of 0.2-5% during stage 5.

  Again, as with cell types from stages 2-4, an increase in expression of genes associated with specific cell types was observed compared to adherent plating controls. For example, increased expression levels of hormones insulin (INS), glucagon (GCG), and somatostatin (SST) were observed. See FIG. 22, Stage I (dl5) of Panel I (INS), Panel J (GCG), and Panel K (SST). Furthermore, the expression levels of genes associated with AFP, ZICl, and ectoderm were significantly reduced in hES-derived cell aggregates compared to adherent plating controls. See FIG. 22, panel M (ZICl) and panel N (AFP) stage 5 (dl5). Thus, directed differentiation that produces pancreatic endocrine cells through cell aggregates in suspension culture removes ectoderm and organ endoderm contaminants.

  Production of hES-derived hormone expressing endocrine population cells was confirmed by flow cytometric analysis at day 23 with the protocol described. The population was initially formed in 5 mL DMEM / F12 at 140 rpm, and alternatively included a knockout serum replacement (KSR; Gibco / Invitrogen, compared to 0063 for consistency) or non-foreign KSR (Invitrogen) Next, it was differentiated at 100 rpm. Analysis of NKX6.1, chromogranin A, insulin, glucagon, and somatostatin protein expression showed that the ES cell-derived population had about 20% NKX6.1 + / chromogranin A-pancreatic epithelium, and about 74% chromogranin A + endocrine tissue. Indicates that it contains. In addition, 11% of the cells express insulin, 14% express glucagon, and 11% express somatostatin. In these, 68% of insulin + cells are single positive, 70% of glucagon + cells are single-positive, and 52% of somatostatin positive cells are single-positive. The degree of single hormone positivity exceeds the value explained in adherent cultures, which were mostly polyhormonal cells.

  To further evaluate the efficiency of assembly differentiation into hormone-expressing endocrine cells, a cryosection of the ES-derived population was analyzed during stage 5 for glucagon, insulin, and somatostatin expression during immunocytochemistry and confocal microscopy. Was investigated using. Image analysis of a 20-fold cold section showed highly efficient differentiation of aggregate cells to hormone-positive, and almost all cells expressed glucagon, somatostatin or insulin. Also, in contrast to previous adhesion culture experiments, it appears that the majority of the cells in the aggregate express a single hormone so that it occurs in vivo during development.

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

  The following shows that the production of hES-derived cell aggregates can be initiated not only from pluripotent hES cells, but cell aggregates were directly initiated and differentiated in differentiation medium (day 0 cell aggregates) It shows that one can start with hES-derived cells, for example, that cell aggregates can be made from stage 1, 2, 4 and 5 or hES-derived cells.

Day 0 cell assembly

Cell aggregate produced on the first day of stage 1 (d0):
Adherent pluripotent hES cells are grown, passaged manually or enzymatically, separated, counted and pelleted, which contains RMPI, pen / strep and glutamax medium, and further 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 It was done. This volume can be increased to> 4 × 10 ⁶ cells / mL but requires 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 about 1-15 microM, 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 equally distributed into each well of the low binding 6 well dish, described above, and placed on a rotating platform at 100 to 140 rpm overnight. During this culturing period, cell aggregates of uniform size and shape were formed. As a result, the high density culture was effectively or substantially enriched for PDX1-positive pancreatic endoderm or PDX-positive pancreatic progenitor cells. Details are provided in Example 21.

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

Stages 1-2 and 3

Cell assemblies produced in stage 1 d2-d3:
Adherent hES cells are grown and passaged substantially as described above, and then substantially as described in d'Amour et al. (2006) (supra). Differentiated to stage 1.

Stage 1 (about d2 or d3 to differentiation protocol;
At the end of the final endoderm-type cells), the adherent culture is washed once with PBS +/− and pre-warmed to 37 ° C. using a 1 mL or 5 mL pipette with 2 mL of Accutase, approximately 2- Separated into single cells for 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. . Cells were counted and pelleted (centrifugated) substantially 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 was cloudy. In some experiments, 5 microM SB431542 (Sigma Aldrich Inc.) or 2.5 microM TGF-beta Inhibitor IV (Calbiochem) was included with KGF. In some experiments, Y27332 (10 microM) was included. The resuspended cells are equally distributed into each well of a low binding 6-well dish as described above and placed on a rotating platform at 100 to 140 rpm overnight to form a cell aggregate of uniform size and shape. It was.

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

Stage 2, 5th to 6th days

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

  Adherent cells at the end of stage 2 (approximately d5 or d6 of the differentiation protocol; foregut type cells) were separated into single cells, counted and pelleted substantially as described above. The cell pellet then contains DMEM, pen / strep and glutamax medium, plus IX B27-supplement and DNase (50-100 microg / ml, Roche Diagnostics) and no FBS 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 Or 2 microM retinoic acid, 0.25 nM KAAD-cyclopamine, and 50 ng / mL noggin (R & D systems); or 0.25 nM KAAD-cyclopamine and 100 ng / mL noggin; or 100 ng / mL noggin; .5μM RA and 0.25n Resuspended in differentiation medium containing either M KAAD-cyclopamine; or 0.2 microM to 0.5 microM RA, 0.25 nM KAAD-cyclopamine and 50 ng / mL noggin. In some experiments, Y27332 (10 microM) was included.

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

  The cell aggregates produced at the end of stage 2 are further differentiated on a rotating platform, fed 1-2X daily, and then on days 0-2, 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 KAAD-cyclopamine and 50 ng / mL noggin (R & D systems); or 0.25 nM KAAD-cyclopamine and 100 ng / mL noggin; or 100 ng / mL noggin; or 0.2 microM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine; or 0 .2 μM to 0.5 μM RA, 0.25 Feeds containing either nM KAAD-cyclopamine and 50 ng / mL noggin were fed.

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

Stages 4 and 5-10 to 30

At stage 4 a cell aggregate was produced at d10-dl4:
Again, adherent hES cells were grown and passaged substantially as described above and then differentiated to stage 2 as described in D'Amour et al. (2006) (supra).

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

  Adherent cultures of stage 4 (about dl0-d14 of differentiation protocol; pancreatic epithelium and endocrine type cells) or stage 5 (about 16 to 30 days of differentiation protocol; endocrine precursors and endocrine cells) are similarly Separated into single cells, counted and pelleted. And cell pellets in pen / strep and glutamax supplemented with DMEM CMRL, and IX B27-supplement, DNase (50-100 microg / ml, Roche Diagnostics), and 0-2% FBS And resuspended. In some experiments, Y27332 (10 microM) was included to support cell survival. Cells were equally distributed into 6-well plates as described above and placed on a rotating platform at 100 to 140 rpm for 4 hours to overnight.

  Furthermore, as in Examples 17-19, the cell aggregates produced in stage 2 and stage 5 were enriched in pancreatic cell types compared to the adherent plate culture from which they were derived. For example, in one typical experiment, cell aggregates produced at stage 2 and analyzed by flow cytometry at stage 4 were at least 98% pancreatic cell type (73% chromogranin A positive endocrine. Cells, 25% Nkx6.1 l positive pancreatic endoderm (PE)), and 2% non-pancreatic cells are of type. The adherent plate from which the cell aggregates are derived consists 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, the aggregates in stage 2 can enrich for progenitor cells resulting in pancreatic cell types and reduce non-pancreatic cell types. Similarly, in a typical experiment, the cell aggregates produced in stage 5 and analyzed by flow cytometry consisted of at least 75% chromogranin A positive endocrine cell types, but the cell aggregates were derived from Adherent plate cultures consist of approximately 25% chromogranin A positive endocrine cell types. Thus, the assembly 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 with cell aggregate suspensions, but also include contaminant populations (eg, ectoderm, trophectoderm, organs Provided are methods for enriching pancreatic cell types (eg, pancreatic endoderm and endocrine cells) while reducing hES-derived pancreatic cell types (or populations) having germ layers and extraembryonic endoderm.

Example 20-Effect of cell density on hES differentiation results

  The following shows that changes in cell density affect differentiation results within specific media and growth factor conditions. The differentiation efficiency resulting from regulation in cell density reflects changes in the concentration of endogenously produced transfer molecules and reflects the concentration dependence of these molecules that affect cell differentiation.

  Human ES cell aggregates, including d0 cell aggregates produced directly in differentiation media, and hES-derived cell aggregates were generated substantially as described above. After 5 days of differentiation via stages 1 and 2, differentiated cell aggregates are pooled and re-aliquoted into individual wells with different seeding densities, eg, 28 mL suspension of foregut endoderm. Stage cell aggregates were seeded or recoated at 4, 6, 8 or 10 mL / well (cell density within 2.5-fold range). This cell distribution was performed twice, and 50 ng / mL in one set of wells with stage 3 medium (DMEM / PenStrep / Glutamax + 1% B27 supplement (vol / vol) +0.25 microM KAAD-cyclopamine + 3 microM TTNPB) Of noggin was supplied, and the other set of wells contained 25 ng / mL noggin. Stage 3 continued for 3 days with daily media changes. Two cell samples were taken for the last 3 days (or about day 8) of stage 3 for real-time QPCR analysis and again after stage 4 (or about day 14).

The cell density and noggin concentration used during stage 3 had different effects on the expression of pancreatic endoderm progenitor cells and / or their genes indicative of endocrine precursor cells or progenitors. Briefly, there is a linear relationship between an increase in cell density and a corresponding increase in pancreatic progenitor cell types (eg, pancreatic endoderm, pancreatic epithelium, PDX1-positive pancreatic endoderm). For example, after stage 3 (or day 8), an increase in cell density corresponds to an increase in the number of pancreatic progenitor cells, as shown by increased gene expression of PDXl and NKX6-1. See Figures 24A and 24B. In contrast, there was an inverse correlation between the increase in cell density and the decrease in endocrine precursor cell type after stage 4 (or day 14).
For example, as cell density decreased, there was a decreased expression of at least NGN3 and NKX2-2 after stage 3 (or day 8). See Figures 24C and 24D.

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

Example 21-Optimization of cell aggregate suspension cultures generated by enrichment of pancreatic endoderm or endocrine cell types

  The cellular composition of the hES-derived cell aggregate population is optimized for a certain cell type by controlling the enrichment of various growth factors and / or drugs. The pancreatic cell compositions described herein are hES cell adhesion cultures, d0 cell aggregates (cell aggregates were initiated from hES adhesion cultures or placed directly in differentiation medium rather than multi-differentiated stem cell medium) , Or as described in previous examples, generated from single cell suspension cultures starting from hES-derived cell aggregate 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 cellular composition of differentiated hES cell aggregates was assessed by flow cytometric analysis using a panel of markers including CHGA, NKX6.1, and PDX1. The total percentage of endocrine cells, pancreatic endoderm cells, PDX1 + endoderm cells, and non-pancreatic cells in any aggregate is shown in Table 4.

  The data in Table 4 shows that by controlling the concentration and ratio of certain growth factors, the resulting composition can be optimized for certain cell types. For example, decreasing the concentration of KGF and EGF (eg, 71% of K (25) E (10) vs 22.1%) increased the proportion of pancreatic endoderm type cells 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 endoderm-type cells, The total number was comparable to endocrine type cells (eg, 39.6% vs. 40.1%). When noggin and KGF at higher concentrations (eg, N (50) K (50)), or growth factors are added, the population of endocrine type cells is compared to the pancreatic endoderm cell type in the resulting aggregate. 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 growth factors. By not adding (1.53%), it can be significantly reduced.

  Thus, Table 4 shows that pancreatic endoderm, endocrine, PDX1-positive endoderm or non-pancreatic cell types can be obtained at least by changing the concentration of different growth factors in the culture medium at a stage of differentiation (eg, stage 4). It is clearly shown that a population is significantly increased and / or decreased.

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

  Methods for enriching hES-derived cells or cell aggregates are not limited to methods using antibody affinity means and are available to those skilled in the art that allow enrichment of certain cell types, or All methods available in the future can be included. Enrichment can be performed 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 generating a cell aggregate suspension described herein is a pancreatic precursor that functions in vivo. To demonstrate providing cells, the hES-derived cell aggregates of Examples 17-21 (eg, PDX1-positive endoderm, pancreatic endoderm, pancreatic epithelium, endocrine precursor, endocrine cells, And the like) were implanted into animals. The method of transplantation into normal and diabetic-induced animals, determination of in vivo glucose responsiveness in animals, and in vivo insulin production of transplanted mature cells were substantially similar to Kroon et al. (2008 ) Nature Biotechnology 26 (4): 443-452, and US patent application Ser. No. 11 / 773,944, filed Jul. 5, 2007, and described in the method of producing pancreatic hormones (METHODS OF PRODUCING PANCREATIC HORMONES). Executed in the 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 Ser. No. 11 / 773,944 (supra).

  The methods, compositions, and devices described herein are representative of preferred embodiments, but are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention and are defined by the scope of the claims. It will be readily apparent to 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 will be understood that in certain aspects related to the invention, combinations of embodiments may be provided even if described as different embodiments. For example, methods for making hES-derived cell aggregates in suspension culture include any endoderm lineage cell type, such as pancreatic lineage type cells, liver lineage type cells, epithelial lineage type cells, thyroid lineage cells, and thymic lineage cells. Made and optimized to produce cells. Thus, it is not limited to the hES-derived cell types specifically described herein. Conversely, although the various aspects of the invention have been described as a single embodiment for the sake of brevity, they can be performed separately or can be provided as any suitable sub-combination. . For example, those skilled in the art will know that hES cells and hES-derived cell aggregates from adherent or suspension cultures, from undifferentiated adherent or suspension cultures, and differentiated adherent plate cultures. Or it is clear that the described methods for producing from suspension cultures are only exemplary and combinations of these methods can also be used.

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

  As used in the following claims and specification, the term “consisting essentially of” is meant to include the elements listed in connection with this phrase, and is disclosed for the elements listed. Limited to other elements that interfere with or do not contribute to the activity or action. That is, the term “consisting essentially of” indicates that the listed element is required and mandatory, but whether it affects the activity and action of the listed element, It means that other elements may optionally be present or cannot be present.

Claims (1)

Primate pluripotent cell-derived cell aggregates suspended in a physiologically acceptable medium.