RU2689710C2 - Suspending and clustering human pluripotent stem cells for their differentiation into pancreatic endocrine cells - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention refers to biotechnology, namely to production of three-dimensional cell clusters and their differentiation. Method involves treatment of pluripotent stem cells, which are induced pluripotent stem cells, cells obtained from human umbilical cord tissue, parthenotes, cells obtained from amniotic fluid, or human embryonic stem cells of line H1, H7, H9, SA002 or BG01v, cultured in a flat adhesive culture together with a chelating agent or enzyme, with release of cell aggregates from a flat adhesive culture. That is followed by suspending cell aggregates from a flat adhesive culture into a culture medium in the presence of a Rho-kinase inhibitor without dissociating cell aggregates to single cells. Then, suspension of cell aggregates is transferred into dynamic suspension culture and increased volume of suspension of cell aggregates in dynamic suspension culture to obtain three-dimensional cell clusters, wherein three-dimensional cell clusters preserve pluripotency. Further, it is possible to differentiate pluripotent cell clusters in a dynamic suspension culture system to obtain a population of precursor cells of pancreatic cells, a population of neuronal progenitor cells or a population of cardiomyocyte precursor cells.EFFECT: invention widens the range of agents.34 cl, 20 tbl, 95 dwg, 16 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a partial continuation of application for US patent No. 13 / 998,974 (filed December 30, 2013), which claims the priority stated in provisional application for US patent No. 61 / 747,799 (filed December 31, 2012) and in the provisional application for US patent No. 61 / 962,158 (filed November 1, 2013), which are fully incorporated herein by reference.

SCOPE OF THE INVENTION

The present invention relates to the field of cell differentiation, including the preparation of embryonic stem cells and other pluripotent cells that support pluripotency in an aggregated cell cluster, in order to differentiate them into endoderm progenitor cells, pancreatic endocrine cells, mesoderm cells or ectoderm cells. In one aspect of the present invention, a method is disclosed for generating pluripotent stem cell clusters and maintaining them in suspension culture to differentiate pancreatic endoterm, pancreatic endocrine progenitor cells and single hormone pancreatic endocrine cells.

BACKGROUND OF THE INVENTION

Recent advances in cell replacement therapy for the treatment of type 1 diabetes and the lack of islets of Langerhans with the possibility of transplantation have led to the development of sources of insulin-producing cells, or β-cells, suitable for transplant engraftment. One approach is to obtain functional beta cells from pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, pluripotent cells give rise to a group of cells containing three germ layers (ectoderm, mesoderm, and endoderm) during a process known as gastrulation. Tissues such as thyroid, thymus, pancreas, intestines and liver tissue will develop from the endoderm through an intermediate stage. The intermediate stage of this process is the formation of definitive endoderm.

By the end of gastrulation, the endoderm is divided into anterior and posterior sections, which can be recognized by the expression of a number of factors that uniquely distinguish the anterior, middle, and posterior regions of the endoderm. For example, HHEX and SOX2 identify the anterior region, while CDX1, 2 and 4 identify the posterior half of the endoderm.

Migration of the endoderm tissue brings the endoderm closer to various mesodermal tissues that contribute to the regionalization of the intestinal tube. This is achieved by a variety of secreted factors, such as FGFs, Wnts, TGF-Bs, retinoic acid (RA), BMP ligands and their antagonists. For example, FGF4 and BMP promote the expression of Cdx2 in the putative endoderm of the hindgut and inhibit the expression of the anterior genes Hhex and SOX2 (Development 2000, 127: 1563-1567). It has been demonstrated that WNT signaling also acts in parallel with FGF signaling, contributing to the development of the posterior intestine and inhibiting the buds of the foregut ( Development 2007, 134: 2207-2217). Finally, retinoic acid secreted by the mesenchyme regulates the border between the anterior and posterior intestine ( Curr Biol 2002, 12: 1215-1220).

The expression level of specific transcription factors can be used to determine the type of tissue. During the conversion of the definitive endoderm into a primitive intestinal tube, the intestinal tube becomes divided into broad domains that can be observed at the molecular level using limited gene expression patterns. For example, a regionalized pancreatic domain in the intestinal tube demonstrates very high PDX1 expression and very low CDX2 and SOX2 expression. The expression level of PDX1, NKX6.1, PTF1A and NKX2.2 is high in pancreatic tissue; and the expression level of CDX2 is high in intestinal tissue.

The formation of the pancreas occurs when the differentiation of the definitive endoderm into the pancreatic endoderm. The dorsal and ventral domains of the pancreas are formed from the epithelium of the anterior intestine. The anterior gut also gives rise to the formation of the esophagus, trachea, lungs, thyroid, stomach, liver, pancreas, and bile duct system.

Pancreatic endoderm cells express pancreato-duodenal, containing the homeobox gene PDX1. In the absence of PDX1, the pancreas does not develop further than the formation of ventral and dorsal buds. Therefore, the expression of PDX1 is an important stage of pancreatic organogenesis. The mature pancreas contains both exocrine and endocrine tissues, which are formed during the differentiation of pancreatic endoderm.

D'Amour et al. describe obtaining enriched cultures of definitive endoderm derived from human embryonic stem cells in the presence of a high concentration of activin and a low serum concentration ( Nature Biotechnol 2005, 23: 1534-1541; US Patent No. 7,704,738). Transplantation of these cells under the kidney capsule in mice resulted in differentiation into more mature cells with endoderm tissue characteristics (US Patent No. 7,704,738). Definitive endoderm cells derived from human embryonic stem cells can be further differentiated into PDX1-positive cells after the addition of FGF10 and retinoic acid (US Patent Application Publication No. 2005 / 0266554A1). The subsequent transplantation of such progenitor cells of pancreatic cells into the fatty body of immunodeficient mice led to the formation of functional pancreatic endocrine cells, followed by a 3-4 month maturation stage (US Patent No. 7,993,920 and US Patent No. 7,534,608).

Fisk et al. Report a system for obtaining pancreatic islet cells from human embryonic stem cells (US Patent No. 7,033,831). Low molecular weight inhibitors have also been used to induce progenitor cells of pancreatic endocrine cells. For example, low molecular weight inhibitors of the TGF-ß receptor and BMP receptors ( Development 2011, 138: 861-871; Diabetes 2011, 60: 239-247) have been used to significantly increase the number of pancreatic endocrine cells. In addition, low molecular weight activators were also used to generate definitive endoderm cells or pancreatic progenitor cell precursors ( Curr Opin Cell Biol 2009, 21: 727-732; Nature Chem Biol 2009, 5: 258-265).

Recently, protocols for the cultivation of progenitor cells, such as pluripotent stem cells, have been greatly improved. PCT Publication No. WO2007 / 026353 (Amit et al.) Discloses techniques for maintaining human embryonic stem cells in an undifferentiated state in a two-dimensional culture. Ludwig et al. , 2006 ( Nature Biotechnology , 24: 185-7) discloses a TeSR1-based medium for cultivating human embryonic stem cells on a matrix. US patent application No. US No. No. 2007/0155013 (Akaike et al.) Describes a method for growing pluripotent stem cells in suspension using a carrier that attaches to pluripotent stem cells, and in US patent application No. US. 2009/0029462 (Beardsley et al) discloses methods for growing pluripotent stem cells in suspension using microcarriers or cell encapsulation. PCT Publication No. WO 2008/015682 (Amit et al.) Describes a method for growing and maintaining human embryonic stem cells in a suspension culture under specific culture conditions in the absence of a substrate substrate.

US patent application No. US No. No. 2008/0159994 (Mantalaris et al. ) Describes a method for cultivating human embryonic stem cells encapsulated in alginate particles in a three-dimensional culture system.

Despite these achievements, a method for successfully cultivating pluripotent stem cells in a three-dimensional culture system, which can differentiate into functional endocrine cells, is still needed.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The foregoing summary of the invention, as well as the subsequent detailed description of the invention, will be better understood when considered together with the attached figures. In order to illustrate the invention, the figures show embodiments of the present invention. It should be understood, however, that the invention is not limited to the exact designs, examples and tools shown.

FIG. 1a shows photomicrographs Dispase ^® treated human embryonic stem cells (hES) cell line H1 immediately after the separation (left panel) and after 24 hours in a non-static attachment culture (right panel) in accordance with Example 1. The cells after separation (left panel) resembled monolayer fragments with an average fragment diameter of approximately 20–30 μm, each fragment consisting of a cluster of cells. After 24 hours, the cells in the non-adherent static culture formed cluster configurations.

FIG. 1b shows the results of flow cytometry for CD9, SSEA4, CXCR4, TRA- 1-60 and TRA-1-81 to Dispase ^® treated human embryonic stem cells from stem line H1 cells after culturing for 4 days in a centrifuge tube at 125 ml, containing 25 ml of mTeSR ^® 1 carrier in accordance with Example 1. The cells showed high expression of pluripotency markers (CD9, SSEA4, TRA-1-60 and TRA-1-81) with almost no CXCR4 expression, a differentiation marker.

FIG. 1c are photomicrographs Dispase ^® treated human embryonic stem cells from stem line H1 cells after 72 and 96 hours at the end of the differentiation stage 1. FIG. 1c, unattached cell aggregates are noticeable after 72 hours at 4x magnification (left panel), 96 hours at 4x magnification (central panel), and 96 hours at 10x magnification (right panel).

FIG. 1d shows the results of flow cytometry Dispase ^® treated human embryonic stem cell lines of stem cells H1 at the end of step 1 differentiation markers CD9, CD184 (CXCR4) and CD99 (see. Example 1). As shown in FIG. 1d, expression of CD9, a marker of pluripotency, almost disappeared, while expression of markers of the final differentiation of the endoderm of CXCR4 (CD184) and CD99 was quite high.

FIG. 1e shows the results of quantitative polymerase chain reaction reverse transcription (qRT-PCR) for expression of selected genes associated with pluripotency and the genes associated with terminal differentiation endoderm treated with Dispase ^® human embryonic stem cells from a line of stem H1 cells at the end of stage 1 in comparison with undifferentiated H1 (WA01) hES cells (see example 1). Cells at the end of stage 1 showed a sharp decrease in gene expression of pluripotency (CD9, NANOG and POU5F1 / OCT4) and increased expression of genes associated with the final differentiation of endoderm (CXCR4, Cerberus (CER1), GSC, Foxa2, GATA4, GATA6, MNX1 and SOX17) , compared to undifferentiated cells of the hES WA01 line.

FIG. 1f shows micrographs Dispase ^® treated human embryonic stem cell lines of stem cells H1, showing further differentiation of the finally differentiated into endoderm, pancreatic endoderm (see. Example 1). As differentiation progresses, morphological changes in cells and cell clusters from stage 2, day 1 (upper left panel) to stage 2, day 3 (upper right panel), then to stage 3, day 4 (lower left panel), and stage 4, day 1 (lower right panel).

FIG. 2a depicts the flow cytometry data of EDTA-treated human embryo cells from the H1 stem cell line after 2 days of culture in a mixed culture suspension treated with EDTA and before proceeding to culture differentiation, for markers associated with pluripotency and differentiation, according to Example 2 The data show high expression of pluripotency markers (CD9, SSEA4, TRA-1-60 and TRA-1-81) with almost no expression of the differentiation marker (CXCR4).

FIG. 2b shows micrographs of EDTA-treated human embryo cells from the H1 stem cell line differentiated at stage 1, day 3 for cells grown in a centrifuge tube and stage 2, day 2, stage 4, day 1 and stage 4, day 3 for cells grown in a centrifuge test tube or Erlenmeyer flask according to example 2. In the suspension of differentiated cultures, essentially uniform and homogeneous populations of cells are formed in spherical aggregates.

FIG. Figure 2c shows the flow cytometry data of EDTA-treated human embryonic stem cells from the H1 stem cell line at the end of stage 1 to cell surface markers showing pluripotency and differentiation of endoderm. As shown in FIG. 2c, expression of CD9, a marker of pluripotency, has almost disappeared, while expression of the marker for the final differentiation of the endoderm of CXCR4 (CD184) was rather high.

FIG. 2d shows the results of qRT-PCR for the expression of selected genes associated with pluripotency and genes associated with the final differentiation of the endoderm of human embryonic stem cells treated with EDTA from the H1 stem cell line at the end of stage 1 compared to undifferentiated H1 (WA01) hES cells (see example 2). FIG. 2d shows a dramatic decrease in the expression of pluripotency genes (CD9, NANOG and POU5F1 / OCT4) and an increase in the expression of genes associated with the final differentiation of endoderm (CXCR4, Cerberus (CER1), Foxa2, GATA4, GATA6, MNX1 and SOX17).

FIG. 2e shows the expression of differentiation markers (Nkx6.1, CDX2, Sox2, and Chromagranin) obtained by flow cytometry of human embryonic stem cells treated with EDTA from the H1 stem cell line, which were differentiated from the beginning of stage 1 into pancreatic endoderm cells by suspending centrifuge tubes or Erlenmeyer flasks according to example 2. Flow cytometry data in both suspension formats show a high level of Nkx6.1, a transcription factor required for functional β-cells ok, and high levels of endocrine pancreas markers, such as synaptophysin (data not shown) and chromogranin.

FIG. 2f shows the results of qRT-PCR expression of individual genes associated with the differentiation of EDTA-treated human embryonic stem cells from the H1 stem cell line, which were differentiated from the beginning of stage 1 into pancreatic endoderm cells by suspending in centrifuge tubes or Erlenmeyer flasks according to Example 2. These data were compared with expression data in the WA01 hES cell line. RT-PCR results show high levels of several pancreatic progenitor genes.

FIG. 3a shows micrographs of human embryonic stem cell line H1, separated by static culture after Accutase ^® processing. As shown in FIG. 3, cells were removed from the surface in the form of small aggregates.

FIG. 3b shows Phase contrast photomicrographs of human embryonic stem cell line H1, separated by static culture after Accutase ^® and after processing grown in suspension culture for three days. FIG. 3b shows the formation of essentially uniform spherical populations of cell clusters.

FIG. 3c is a photomicrograph of human embryonic stem cell line H1, separated by static culture after Accutase ^® treatment, grown in suspension culture for three days and have been subjected to serial passirovke using Accutase ^® dissociation.

FIG. Figure 4a shows micrographs of human embryonic stem cells of the H1 line, brought to a suspension culture state using various protocols of directional differentiation at various stages of differentiation. FIG. 4 shows micrographs of cells at each stage of differentiation.

FIG. 4b shows flow cytometry results for differentiation markers (CXCR4, CD56 and Pdx1) of human embryo stem cells of the H1 line brought into suspension culture using different protocols of differentiation at different stages of differentiation (hours after differentiation began). At the end of the process of differentiation on the 4th day of the 4th stage, a high percentage of cells showed positive expression of Pdx1.

FIG. 4c shows glucose levels in fed SCID-Bg mice, which were encapsulated differentiated cells implanted with TheraCyte ™.

FIG. 5a shows the results obtained by flow cytometry of EDTA-treated human embryonic stem cells from the H1 stem cell line prior to the start of the differentiation process into cell surface markers, showing pluripotency and differentiation of endoderm. As shown in FIG. 5a, high expression of CD9, SSEA4, TRA-1-60 and TRA-1-80 pluripotency markers was observed.

FIG. 5b shows a phase contrast image of the cells and flow cytometry results of the CXCR4 / CD184 and CD99 differentiation markers, as well as the CD9 pluripotency marker with three different feed settings during stage 1. Testing was performed under the following conditions: without changing the medium after 48 hours; (B) replace the medium 24 hours after the start of differentiation and add a bolus of glucose after 48 hours; and (C) without changing the medium during stage 1 with the addition of glucose bolus and GDF8 24 hours after the start of differentiation and the subsequent addition of glucose bolus 48 hours after the start.

FIG. 5c shows phase-contrast images of differentiated cells exhibiting pancreatic endoderm morphology, which were differentiated using the following feed settings during definitive endoderm formation: (A) medium change 24 hours after differentiation began, 48 hours after medium change (B) replace the medium 24 hours after the start of differentiation and add a bolus of glucose after 48 hours; and (C) without changing the medium during stage 1 with the addition of glucose bolus and GDF8 24 hours after the start of differentiation and the subsequent addition of glucose bolus 48 hours after the start.

FIG. 5d shows flow cytometry results for individual pancreatic (Nkx6.1 and chromagranin) gene expression markers and selected non-pancreatic genes (CDX2 and Sox2) of the differentiated cell at the end of stage 4, which were differentiated using the following feed settings for generating definitive endoderm: (A) replacement of the medium 24 hours after the start of differentiation, without changing the medium after 48 hours; (B) replace the medium 24 hours after the start of differentiation and add a bolus of glucose after 48 hours; and (C) without changing the medium during stage 1 with the addition of glucose bolus and GDF8 24 hours after the start of differentiation and the subsequent addition of glucose bolus 48 hours after the start.

FIG. 5e shows the results of qRT-PCR of the selected expression of pancreatic genes and non-pancreatic genes for differentiated cells at the end of the 4th stage, which were differentiated using the following feed settings during the formation of definitive endoderm: (A) medium change 24 hours after differentiation began without changing the medium after 48 hours; (B) replace the medium 24 hours after the start of differentiation and add a bolus of glucose after 48 hours; and (C) without changing the medium during stage 1 with the addition of glucose bolus and GDF8 24 hours after the start of differentiation and the subsequent addition of glucose bolus 48 hours after the start. Data is shown as a multiple of the difference in expression relative to undifferentiated H1 cell line (WA01) (initial expression 1).

FIG. 5f shows the expression of C-peptide in SCID-Bg mice with implanted cells differentiated according to condition A (change of medium 24 hours after the start of differentiation, without change of medium after 48 hours). Each SCID-Bg mouse implanted 5 million cells under the renal capsule. As shown in FIG. 5f, from 12 weeks after implantation, human C-peptide was detected at levels above 1 ng / ml, and after 16 weeks the levels of C-peptide averaged 2.5 ng / ml.

FIG. 5g shows the effect of glucose treatment on selected SCID-BG mice before and after the introduction (for example, implantation) of cells differentiated according to condition A (change of medium 24 hours after the start of differentiation without changing the medium after 48 hours). As shown in FIG. 5g, glucose treatment caused a significant increase in the circulating human C-peptide on average from 0.93 ng / ml in the hungry state to 2.39 ng / ml in the saturated state.

FIG. 5h shows the effect of administration of streptozotocin (STZ) (i.e. STZ-induced diabetes) to SCID-BG mice that were injected with cells differentiated according to condition A (change of medium 24 hours after the start of differentiation, without changing the medium after 48 hours). As shown in FIG. 5h, animals with a graft of functional GSIS-competent tissue (i.e., one into which cells were introduced) maintain a normal level of blood glucose, unlike the untreated control group, which developed frank diabetes.

FIG. 6a shows micrographs of human embryonic stem cells of the H1 line grown on three spheres of Cytodex ^® microcarrier prior to differentiation.

FIG. 6b shows micrographs of human H1 embryo stem cells grown on three spheres of Cytodex ^® microcarrier at various stages of differentiation.

FIG. 6c shows the number of cells (cells / cm ² ) depending on the day of differentiation for human embryo stem cells of the H1 line grown and differentiated on media in media containing activin A (AA) and WNT3A (media WTN3A / AA), microcarriers in medium, containing activin A and WNT3a (microcarriers WTN3A / AA), media in a medium containing MCX and GDF8 (MCX / GDF8 carriers) and microcarriers in a medium containing MCX and GDF8 (MCX / GDF8 microcarriers).

FIG. 6d shows the number of cells (cells / ml) depending on the day of differentiation for human embryo stem cells of the H1 line grown and differentiated on carriers in media containing activin A and WNT3A (media WTN3A / AA), microcarriers in media containing activin A and WNT3a (microcarriers WTN3A / AA), media in a medium containing MCX and GDF8 (MCX / GDF8 media) and microcarriers in a medium containing MCX and GDF8 (MCX / GDF8 microcarriers).

FIG. 6e shows the results of flow cytometry for the first stage of differentiation of cells grown on a microcarrier culture or a flat culture in the presence of: (a) WNT3A and AA; or (2) MCX and GDF8 as a scatter plot of cell expression of CXCR4 / CD184 (Y axis) and CD9 (X axis).

FIG. 6f shows flow cytometry results for the first stage of differentiation of cells grown on a microcarrier culture or a flat culture in the presence of: (a) WNT3A and AA; or (2) MCX and GDF8 in the total expression of each of the markers (CXCR4 and CD9).

FIG. 6g shows the results of quantitative PCR expression of selected genes associated with the differentiation of human embryonic stem cells of the H1 line, which were differentiated by growing on a flat culture or microcarrier spheres in suspension culture in the presence of: (a) WNT3A and AA; or (2) MCX and GDF8.

FIG. 7 shows the number of cells at different stages of differentiation in the bioreactor from stage 1, day 1 to stage 4, 3 days for cells differentiated according to the protocol of example 7. The number of cells is shown in million cells / ml, as determined by means of a cytometer images (NucleoCounter ^® ).

FIG. 8 shows the average daily pH levels of the bioreactor as a function of time (differentiation day) according to the differentiation protocol of Example 7. The pH levels were determined using NOVA BioProfile ^® FLEX (Nova Biomedical Corporation, Waltham, Mass. USA).

FIG. 9 shows average levels of lactate medium in the bioreactor as a function of time (days of differentiation), according to the protocol of Example 7. differentiation lactate levels were determined by ^{NOVA BioProfile ® FLEX (Nova Biomedical Corporation} , Waltham, Massachusetts, USA).

FIG. 10 shows the average level of glucose in the medium in the bioreactor as a function of time (days of differentiation) according to the protocol of Example 7. differentiation glucose levels were determined by ^{NOVA BioProfile ® FLEX (Nova Biomedical Corporation} , Waltham, Massachusetts, USA).

11 shows undifferentiated gene expression, determined by the QRT-PCR method, for stage 0, 1 day (i.e. twenty four hours after seeding), the cells are differentiated according to the protocol of example 7 for the array of pluripotency that contains the selected genes, associated with pluripotency.

FIG. 12 shows undifferentiated gene expression, as determined by the QRT-PCR method, for stage 0, 1 day (ie, twenty-four hours after seeding) cells for the definitive endoderm (“DE”) of the array, which contains the selected genes associated with the definitive endoderm (Example 7 ).

Fig. 13 shows undifferentiated gene expression, determined by the QRT-PCR method, for a stage of 0, 3 days (i.e. seventy-two hours after seeding cells) for an array of pluripotency that contains selected genes associated with pluripotency (see Example 7).

FIG. 14 shows undifferentiated gene expression, as determined by the QRT-PCR method, for a stage of 0, 3 days (i.e. seventy-two hours after seeding) cells for a DE array that contains selected genes associated with DE (see Example 7).

FIG. 15 shows the results of fluorescent activated cell sorting (FACS) for CD9, CD184 / CXCR4, SSEA4, TRA-1-60 and TRA-1-81 for undifferentiated stage 0, day 3 (ie, seventy-two hours after seeding) of cells ( see example 7). The results are also shown in Table 8.

FIG. 16 shows undifferentiated gene expression, as determined by the QRT-PCR method, for some genes of stage 0, 1 day (that is, twenty-four hours after seeding) and stage 0, day 3 (ie, seventy-two hours after seeding), the cells are differentiated into according to the protocol of Example 7. In particular, in FIG. 16 shows a slight increase in gene expression for GATA4, RCG, MIXL1 and T and an increase in ≥100x of GATA2 expression during stage 0 prior to directed differentiation.

FIG. 17 shows the undifferentiated expression of the gene, as determined by the QRT-PCR method, for the DE array, which contains the selected genes associated with the DE, for stage 0, 1 day (that is, twenty four hours after seeding) and stage 0, day 3 (ie seventy-two hours after seeding differentiated cells) in accordance with the protocol of Example 7. In particular, FIG. 17 shows an increase of ≥100x CER1, FGF17 and the expression of FGF4 during stage 0 before directional differentiation.

FIG. 18 and 19 show the expression of genes for stage 1, day 1 of cells differentiated according to the protocol of example 7. FIG. 18 shows the gene expression determined by the qRT-PCR method for an array of pluripotency that contains selected genes associated with pluripotency for stage 1, day 1 of the cell. FIG. 19 shows the gene expression determined by the qRT-PCR method for the DE array, which contains the selected DE genes, for stage 1, day 1 of the cell. FIG. 18 and 19 illustrate significant changes in the expression structure of genes, such as a ~ 700x increase in Foxa2 expression and a 1000x increase in expression of CER1, EOMES, FGF17, FGF4, GATA4, GATA6, GSC, MIXL1 and T.

FIG. 20 and 21 show gene expression for stage 1, day 3 of cells differentiated according to the protocol of example 7. FIG. 20 shows the gene expression determined by the qRT-PCR method for an array of pluripotency that contains selected genes associated with pluripotency, for stage 1, day 3 cells. FIG. 21 shows the gene expression determined by the QRT-PCR method for the DE array, which contains the selected DE genes, for stage 1, day 3 cells.

FIG. Figure 22 shows the FACS results for CD9, CD184 (also known as CXCR4) and CD99 in stage 1, day 3 cells differentiated according to the protocol of Example 7. There was an almost complete transition from the CD9-expressing / CXCR4-negative population of iPS cells when initiating differentiation (Fig. 15) to a homogeneous population of cells expressing CXCR4 (98.3% of CXCR4-positive cells, ± 1.9 SD) at the end of stage 1 (Fig. 22).

FIG. 23 shows the expression of genes determined by the qRT-PCR method for the DE array, which contains the selected genes associated with the DE for stage 1, day 3; stage 2, day 1; and stage 2, day 3 for cells differentiated according to the protocol of example 7. FIG. 23 shows that the levels of expression of HNF4α and GATA6 at stage 2, days 1 and 3, increase, while genes with a high level of expression at day 3 of stage 1 (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX17 and FV) showed a decrease in expression at the end of stage 2.

FIG. 24 shows the expression of the AFP, Pdx1 and Prox1 genes of the anterior intestine, as determined by the QRT-PCR method, for stage 2, day 1 of cells and stage 2, day 3 of cells differentiated according to the protocol of example 7. As shown in FIG. 24, the expression of these genes increased.

FIG. 25 shows the FACS results for Pdx1, Foxa2, chromogranin, Nkx2.2 and SOX2 for stage 3, day 3 cells grown in stage 3 medium (Table 7) and differentiated according to the protocol of Example 7. As shown in FIG. 25, cell expression markers correspond to the endoderm of the pancreatic line, as determined by the expression of PDX1 and FOXA2 (90.9% ± 11.9 CO of PDX1 positive and 99.2% ± 0.6 CO of FOXA2 positive).

FIG. 26 shows the gene expression determined by the QRT-PCR method, for stage 4 of the array, which contains the selected genes associated with the 4th stage, at stage 3, 1 day and 3 stages, 3 cells per day, differentiated according to the protocol of example 7, FIG. 26 shows that these cells have an increased level of genes usually expressing in the pancreas (ARX, GAST, GCG, INS, Isl1, NEUROD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, PAX6, Ptf1a and SST).

FIG. 27 shows the FACS results for Nkx6.1, Chromagranin (CHGA), CDX2, Sox2, Nkx2.2, Pdx1, Foxa2 and NeuroD for stage 4, day 3 cells differentiated according to the protocol of Example 7. As shown in FIG. 27, at stage 4, day 3, cells maintain high levels of expression of PDX1 and FOXA2, and further, an expression pattern appears corresponding to a combination of pancreatic endocrine cells (28.1% ± 12.5 WITH chromogranin-positive) and pancreatic progenitor cells (58.3 % ± 9.7 WITH NKX6.1-positive.

FIG. 28 shows the expression of genes determined by the qRT-PCR method, for stage 4 of the array, which contains the selected genes associated with the 4th stage, at stage 3, day 3; stage 4, day 1 and stage 4, day 3 for cells differentiated according to the protocol of example 7. FIG. 28 shows an increased level of expression of genes commonly expressed in the pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST).

FIG. 29 shows average FACS results for Nkx6.1, chromagranin (CHGA), CDX2, Sox2, Nkx2.2, Pdx1, Foxa2 and NeuroD for stage 4, day 3 cells differentiated according to the protocol of example 7. In particular, FIG. 29 shows the average FACS expression pattern of the progenitor cells of pancreatic cells generated in a 3L scale from a different seed lot.

FIG. 30 shows the average FACS results for Nkx6.1, chromagranin (CHGA), CDX2, Sox2, Nkx2.2, Pdx1, Foxa2 and NeuroD for stage 4, day 3 cells differentiated according to the protocol of example 7. Before differentiation in stage 4, For 3 days, the cells multiplied to form an ISM, and then grown in stage 0, either in the laboratory-developed “IH3” medium or in the Essential8 ™ medium, to each of which 0.5% BSA was added. Cells grown in medium IH3 are called “IH3-P-grown cells”, and cells grown in Essential8 ™ are called “EZ8-grown cells”. There was no significant difference in the expression pattern in cells grown in different media.

FIG. 31 shows the average FACS results for Nkx6.1, Chromagranin (CHGA), CDX2, SOX2, Nkx2.2, Pdx1, Foxa2 and NeuroD for stage 4, day 3 cells that were previously grown at different pH levels in stage 0 (Example 7 ). No significant changes were observed in the cell profile of stage 4, day 3.

FIG. 32 compares FACS results for Nkx6.1, Chromogranin (CHGA), CDX2, SOX2, Nkx2.2, Pdx1, Foxa2 and NeuroD for stage 4, day 3 cells that were not treated with antifoam-C, and stage 4, day 3 cells that have been treated with an antifoam-C emulsion (94 ppm) (see example 7). The emulsion defoamer-C (Sigma catalog number A8011) showed no effect on the profile of the stage 4 days 3 cells.

Figures 33 to 35 show gene expression as determined by the QRT-PCR method for individual cell genes differentiated according to the protocol of Example 8. FIG. 33 shows gene expression as determined by the qRT-PCR method for some genes in cells twenty-four hours before differentiation begins (see example 8). As shown in FIG. 33, cells from the bioreactor retain gene expression characteristic of pluripotency (POU5F1, NANOG, Sox2 and ZFP42) and show minimal or absent induction of differentiation-specific genes (AFP and Foxa2: <50-fold increase; FOXD3, GATA2, GATA4, GSC , HAND2, MIXL1 and T: <10-fold increase in expression). FIG. 34 shows the gene expression determined by the qRT-PCR method for individual cell genes twenty-four hours after the start of differentiation. FIG. 35 shows gene expression as determined by the qRT-PCR method for individual cell genes seventy-two hours after the start of differentiation.

Figures 36 (a) -36 (e) show the expression of a gene as determined by the qRT-PCR method for individual cell genes differentiated in steps 2-3 and 4 according to the protocol of example 8. In particular, these figures show the expression of genes in cells at stage 2, day 1; stage 2, day 2; stage 2, day 3; stage 3, day 3; and, depending on the gene, stage 4, day 1. FIG. 36 (a) shows the expression of the alpha-fetoprotein gene, Atoh1 and CDX2. FIG. 36 (b) shows the expression of the GAST Hand1, HHEX and HNF4a genes. FIG. 36 (c) shows the expression of the Nkx2.2, Nkx6.1 gene, OSR1 and Pdx1. FIG. 36 (d) shows the expression of Prox1, PFT1a, SOX17 and SOX2 genes. FIG. 36 (e) shows the expression of the SOX9 gene. The data are shown as the difference in expression relative to undifferentiated H1 (WA01) HES cell line (initial expression 1).

FIG. 37 shows gene expression as determined by the QRT-PCR method for individual cell genes in step 4, day 3 of differentiation according to the protocol in Example 8. As shown in FIG. 37, at the end of differentiation in stage 3, day 3 cells differentiated into pancreatic progenitor cells characterized by a high expression level of Pdx1 (> 1 × 10 ⁶ times induction) and other pancreatic genes (> 1000 times induction of ARX, GCG, GAST, INS, ISL, NEUROD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Ptf1a, and SST) and an almost complete loss of OCT4 / POU5F1 expression compared to the undifferentiated stem cells of the H1 human embryo.

FIG. 38 shows the counting of cells daily during the differentiation protocol of Example 8. In particular, Fig. 38 shows the density of cells depending on the day of the process. FIG. 38 shows the number of cells for differentiation of the protocols in the two runs of the reactors (PRD1205 and PRD1207) conducted at pH 6.8 and 7.2. For comparison, cell drift cell counts are also shown.

FIG. 39 (a) - FIG. 39 (d) illustrate the in vivo biological activity of stage 4 day 3 cells differentiated according to the protocol of example 8 and implanted into SCID-BG mice. Cells were implanted subcutaneously using a TheraCyte ™ device under the renal capsule or implanted after incubation on ultra-small carriers. Mice were tested for glucose and C-peptide in the blood every four weeks after implantation of the graft. FIG. 39 (a) shows C-peptide levels after implantation of 5 × 10⁶ or 10 × 10⁶ cells at stage 4 day 3 in a TheraCyte ™ device depending on time. FIG. 39 (b) shows glucose levels in fed animals after implantation of 5 × 10⁶ or 10 × 10⁶ cells at stage 4 day 3 in a TheraCyte ™ device. The mice in FIG. 39 (b) were treated with STZ to ablate the function of β-host cells prior to implantation. FIG. 39 (c) shows the level of C-peptide obtained after implantation of previously cryopreserved cells at stage 4 day 3 in a TheraCyte ™ device depending on the time (week after implantation). FIG. 39 (d) compares the C-peptide levels of mice that received a renal graft of non-cryopreserved / fresh cells of stage 4, day 3 or cryopreserved cells of stage 4, day 3, implanted immediately after thawing (D0) or 1 day after thawing (D1).

FIG. 40A-40D show expression graphs of CXCR4, CD99 and CD9 cells differentiated for three days according to the protocol of example 9, which were processed in step 1, day 1 with the following: MCX-compound and GDF-8 (Fig. 40A); only MCX (Fig. 40B); WNT3A and activin A (Fig. 40C); and only WNT3A (Fig. 40D). These figures show that in suspension culture the addition of 3 μM MCX in the absence of substances of the TGF-β family for one day of differentiation generates a definitive endoderm at a level comparable to that obtained by treating cells with 3 μM MCX plus 100 ng / ml GDF-8 or 20 ng / ml WNT-3A plus 100 ng / ml activin in one day.

FIG. 41A-41D show graphs of the expression of CXCR4, CD99, CD9 and differentiated cells for three days in accordance with the protocol of Example 10, treated with different amounts of MCX in stage 1, day 1. In particular, the cells in stage 1, day 1 were treated: 4 μmol MCX (Fig. 41A); 3 μM MCX (FIG. 41B); 2 μM MCX (FIG. 41C); and 1.5 μM MCX (FIG. 41D).

FIG. 42A and 42B show FACS plots for CXCR4, CD99, and CD9 cells differentiated for three days in accordance with the protocol of Example 11. In particular, these figures show the role of the medium replacement rate in suspension culture. FIG. 42A graphs of the expression of CXCR4, CD99, CD9 and differentiated cells for three days in accordance with the protocol of example 10, with a complete replacement of the medium in step 1. FIG. 42B graphs of expression of CXCR4, CD99, CD9 and differentiated cells for three days in accordance with the protocol of example 10 without changing the medium on day 3. These data indicate that in the suspension culture system, in those cultures that receive a medium change for the third day ( figa) differentiation, resulting in the definitive endoderm appears the same as in cultures that do not receive a three-day medium change (fig.42b).

FIG. 43A and 43B show graphs of the expression of CXCR4, CD99, CD9 and differentiated cells for three days in accordance with the protocol of Example 12. In particular, these figures show the role of GlutaMAX ™ in suspension culture. Cells were cultured in stage 1 in medium supplemented with 1 × GlutaMAX ™ (Fig. 43A) with or without GlutaMAX ™ or without any glutamine (0 M GlutaMAX ™) (Fig. 43B). These data suggest that in the suspension culture system, the addition of GlutaMAX ™ does not affect the efficiency with which the definitive endoderm is formed.

Figures 44A to 44D show the effect of varying amounts of sodium bicarbonate on cells differentiated according to the protocol of Example 13. FIG. 44A and 44B show graphs of the expression of CXCR4, CD99, CD9 and differentiated cells for three days in accordance with the protocol of example 13 with the introduction of 3.64 g / l (FIG. 44A) or 2.49 g / l (FIG. 44B) on stages 1. Figures 44C and 44D of FIG. show phase-contrast micrographs of differentiated cells for three days in accordance with the protocol of example 13 with the introduction of 3.64 g / l (Fig. 44C) or 2.49 g / l (Fig. 44D) on stage 1.

FIG. 45 shows the daily number of cells for cell density, depending on cell differentiation, differentiated according to the protocol of Example 14. The number of cells was determined by cytometer based on images (NucleoCounter ^®).

FIG. 46 shows average daily pH levels in a bioreactor as a function of time (differentiation day) according to the differentiation protocol of Example 14. The pH levels were determined using NOVA BioProfile ^® FLEX (Nova Biomedical Corporation, Waltham, Mass. USA).

FIG. 47 shows the average level of glucose in the medium in the bioreactor as a function of time (days of differentiation) according to the protocol of Example 14. differentiation glucose levels were determined by ^{NOVA BioProfile ® FLEX (Nova Biomedical Corporation} , Waltham, Massachusetts, USA).

FIG. 48 shows the average daily lactate level of the medium in the bioreactor as a function of time (day of differentiation), according to the differentiation protocol of example 14. Lactate levels were determined using NOVA BioProfile ^® FLEX (Nova Biomedical Corporation, Waltham, Mass. USA).

FIG. 49 shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for an array of pluripotency, which contains selected genes associated with pluripotency, for stage 0, 1-3 days and stage 1, 1-3 days of cells, differentiated according to the protocol of example 14.

FIG. 50 shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for a DE array that contains selected genes associated with DE for stage 0, 1-3 days, stage 1, 1-3 days and stage 2, day 1-3 cells, differentiated in accordance with the protocol of example 14.

FIG. 51 shows the FACS results for markers associated with pluripotency (CD184 / CXCR4, SSEA4, TRA-1-60 and TRA-1-81) for stage 0 cells prior to differentiation according to the protocol of Example 14. In particular, FIG. 51 shows high expression of markers associated with pluripotency.

FIG. 52 shows plots of expression of markers of the definitive endoderm of CXCR4, CD99, CD9 and differentiated cells at the end of stage 1 according to the protocol of Example 14.

FIG. 53 shows gene expression as determined by the qRT-PCR method in multiple expression as compared to undifferentiated cells for GAPDH, AFP, HHEX, HNF4α, PDX1 and PROX1 for stage 2, day 1; stage 2 day 2 and stage 2, day 3 for cells differentiated according to the protocol of example 14. FIG. 53 shows an increase in the expression of genes in the foregut (AFP, HHEX, Pdx1 and Prox1).

FIG. 54 shows the gene expression determined by the qRT-PCR method in multiple terms as compared to undifferentiated cells for GAPDH, AFP, CDX2, GAST, HNF4A, NKX2-2, OSR1, Pdx1 and PFT1A for stage 2, day 1-3 days and 3, day 1-3 cells differentiated according to the protocol of Example 14. As shown in FIG. 54, Pdx1 expression increased 60-fold, from 12,000x relative to control at the end of the stage for 2, 3 days and to 739,000x relative to control at the end of the stage for 3, 3 days

FIG. 55 shows gene expression as determined by the method of qRT-PCR, in multiple terms as compared to undifferentiated cells for some genes of stage 3, day 1 to stage 3 and stage 4, day 1-3 cells differentiated according to the protocol of example 14. the top panel in FIG. 55 shows the expression of the genes GAPDH, AFP, ALB, ARX, CDX2, CHGA, GAST, GCG, IAAP, INS, Isl1 and MAFB. Bottom panel of FIG. 55 shows gene expression of MAFB, MUCS, NEUROD1, NEUROG3, NKX2-2, NKX6-1, Pax4, Pdx1, POUSF1, Ptf1a, SST and ZlC1.

FIG. 56 shows micrographs of the final stage of cells differentiated according to the protocol of Example 14. As shown in FIG. 56 representative micrographs (4x) of cell clusters at stage 0 and at the end of differentiation in stages 1 through 4.

FIG. from 57 to 80 show gene expression, determined by the qRT-PCR method, in multiple terms as compared to undifferentiated cells, in cells differentiated according to various embodiments of the protocol of example 15 after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation for the following genes: AFP (Fig. 57); CD99 (FIG. 58); CD9 (Fig. 59); CDH1 (FIG. 60); CDH2 (FIG. 61); CDX2 (Fig. 62); CER1 (Fig. 63); CXCR4 (FIG. 64); FGF17 (FIG. 65); FGF4 (FIG. 66); FOXA (Fig. 67); GADPH (Fig. 68); GATA4 (Fig. 69); GATA6 (Fig. 70); GSC (Fig. 71); KIT (Fig. 72); MIXL1 (Fig. 73); MNX1 (FIG. 74); NANOG (Fig. 75); OTX2 (Fig. 76); POUF5F1 (Fig. 77); SOX17 (FIG. 78); SOX7 (Fig. 79) and T (Fig. 80).

FIG. 81 shows the percentage of cells in the G0 / G1 cell cycle stages for cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15. In particular, FIG. 81 shows the results for the clusters that were processed on the first day of differentiation with one of six conditions: (1) pure, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), ( 3) 3 μmol MCX only, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (catalog number 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml WNT-3A only.

FIG. 82 shows the effects of EDU treatment of cell clusters differentiated according to the protocol of Example 15. The left panel shows the percentage of cells in the G2 / M cell cycle stages for cells after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation in According to various embodiments of the protocol of example 15. In particular, the left panel shows the results for clusters that were treated on the first day of differentiation according to one of six conditions: (1) clean, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), (3) 3 µM Mole MCX only, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (Catalog No. 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml of WNT-3A only. In one data set, these clusters also processed EDUs. In the right pane of FIG. 82 shows the% of EDU-positive cells after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15.

FIG. 83 shows the general operational parameters used in the protocols of Example 15.

FIG. 84 shows the number of EDU inclusions in cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15. In particular, in FIG. 84 shows the results of EDU-incubated cell clusters that were processed on the first day of differentiation according to one of six conditions: (1) pure, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech ), (3) 3 μM only MCX, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (Catalog No. 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (Catalog No. 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml only WNT-3A.

FIG. 85 shows the percentage of cells in the G0 / G1 cell cycle stages for cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15. In particular, FIG. 85 shows the results for clusters that were processed on the first day of differentiation with one of six conditions: (1) pure, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), ( 3) 3 μmol MCX only, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (catalog number 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml WNT-3A only.

FIG. 86 shows the percentage of cells in the S-phase of the cell cycle for cells after 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15. In particular, FIG. 86 shows the results for clusters that were processed on the first day of differentiation with one of six conditions: (1) pure, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), ( 3) 3 μmol MCX only, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (catalog number 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml WNT-3A only.

FIG. 87 shows the percentage of cells in the S-phase of the cell cycle for cells after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation in accordance with various embodiments of the protocol of Example 15. In particular, FIG. 87 shows the results for clusters that were processed on the first day of differentiation with one of six conditions: (1) pure, (2) 3 μM MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), ( 3) 3 μmol MCX only, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (catalog number 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml WNT-3A only.

FIG. 88A - 88E show the expression of a gene determined by the method of qRT-PCR, in multiple terms as compared to undifferentiated cells for cells differentiated according to various embodiments of the protocol of example 15 after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation. FIG. 88A shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for CD99, CD9, CDH1 and CDH2. FIG. 88A shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for CXD2, CER1, CXCR4 and FGF17. FIG. 88C shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for FGF4, FOXA, GATA4 and GATA6. FIG. 88D shows gene expression as determined by the qRT-PCR method, in multiple terms, compared with undifferentiated cells for GSC, KIT, MIXL1 and MNX1. FIG. 88E shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for NANOG, Otx2, POUF5F1 and SOX17. FIG. 88F shows gene expression as determined by the qRT-PCR method, in multiple terms, compared to undifferentiated cells for SOX7 and T. Basic data for figures 88A-88F are shown in FIG. 58-67 and 69-80.

FIG. 89 shows the gene expression patterns of pluripotent cells as determined by the QRT-PCR method cultured in the medium for differentiation of the ectoderm in accordance with the protocol of Example 16. As shown in FIG. 89, cells differentiate towards neuronal cell lines. In particular, the left panel of FIG. 89 shows gene expression patterns for a line of induced pluripotent stem cells formed from umbilical cord tissue cells (UTC). The right panel of FIG. 89 shows gene expression patterns for the sub-clone WB0106 of the H1 HES cell line.

FIG. 90 shows the expression patterns of pluripotent cell genes cultured in mesodermal differentiation media as determined by the qRT-PCR method according to the protocol of Example 16. As shown in FIG. 90, the cells differentiate towards the cells of the heart line. In particular, the left panel of FIG. 90 shows gene expression patterns for a line of induced pluripotent stem cells formed from umbilical cord tissue cells (UTC). The right panel of FIG. 90 shows gene expression patterns for the sub-clone WB0106 of the H1 HES cell line.

FIG. 91 shows the gene expression patterns of pluripotent cells, as determined by the QRT-PCR method, cultured in ectoderm differentiation medium according to the protocol of Example 16. As shown in FIG. 91, the cells differentiate towards the neural line cells. In particular, the left panel of FIG. 91 shows gene expression patterns for a line of induced pluripotent stem cells formed from umbilical cord tissue cells (UTC). The right panel of FIG. 91 shows gene expression patterns for the sub-clone WB0106 of the H1 HES cell line.

FIG. 92 shows protein expression patterns for PAX6, Sox2 and POU5F1 / OCT4, as determined by FACS, pluripotent cells cultured for three days in an ectoderm differentiation medium in accordance with the protocol of Example 16. In particular, the left panels of FIG. 92 shows the expression patterns of Pax6, Sox2 and POU5F1 / OCT4 for a line of induced pluripotent stem cells formed from cells of umbilical cord tissue (UTC). The right panel in FIG. 92 shows protein expression patterns for PAX6, Sox2, and POU5F1 / OCT4 for sub-clone WB0106 of the H1 HES cell line.

FIG. 93 shows the expression patterns of pluripotent cell genes cultured in mesodermal differentiation media as determined by the QRT-PCR method according to the protocol of Example 16. As shown in FIG. 93, the cells differentiate towards the cells of the heart line. In particular, the left panel of FIG. 93 shows gene expression patterns for a line of induced pluripotent stem cells formed from umbilical cord tissue cells (UTC). The right panel of FIG. 93 shows gene expression patterns for the sub-clone WB0106 of the H1 HES cell line.

FIG. 94 shows micrographs of cells differentiated in the mesoderm differentiation medium according to the protocol of Example 16. As shown in FIG. 94, the cells differentiate towards the cells of the heart line. In particular, the left panels of FIG. 94 show micrographs of cells of the sub-clone WB0106 of the H1 HES cell line for 3 days, 5 days and 10 days of differentiation. Right panel of FIG. 94 shows a photomicrograph of a line of induced pluripotent stem cells formed from umbilical cord tissue cells (UTC IPSCs) after 10 days of differentiation.

FIG. 95 shows micrographs of cells differentiated in the ectoderm differentiation medium according to the protocol of Example 16. As shown in FIG. 95, the cells differentiate towards the neural line cells. In particular, the left panels of FIG. 95 show micrographs of cells of the sub-clone WB0106 of the H1 HES cell line for 3 days, 5 days and 10 days of differentiation. Right panel of FIG. 95 shows a photomicrograph of a line of induced pluripotent stem cells derived from umbilical cord tissue cells (UTC iPCS) after 10 days of differentiation.

DETAILED DESCRIPTION OF THE INVENTION

This application is devoted to the preparation of embryonic stem cells and other pluripotent cells that maintain pluripotency in an aggregated cell cluster for differentiation into endoderm progenitor cells, pancreatic endocrine cells, mesoderm cells or ectoderm cells. For clarity of description, and not to limit the invention, the detailed description of the present invention is divided into the following subsections, describing or illustrating certain features, embodiments or uses of the present invention.

Definitions

Stem cells are undifferentiated cells, defined as having the ability to self-renew and differentiate at the single-cell level. Stem cells can produce progeny cells, including self-renewing progenitor cells, non-renewing progenitor cells, and finally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of different differentiation lines from a variety of germ layers (endoderm, mesoderm, and ectoderm). Stem cells also give rise to tissues of multiple germ layers after transplantation and make a significant contribution to the formation of most, if not all, tissues after injection into the blastocyst.

Stem cells are classified by developmental potential. “Cell culture” or “culture” essentially refers to cells taken from a living organism and grown under controlled conditions (“in culture” or “cultured”). Primary cell culture refers to a culture of cells, tissues, or organs taken directly from the body (s) prior to the first reseeding. Cells are propagated in culture when they are placed in a growth medium under conditions that facilitate one or both of the growth and division of cells, which leads to a larger population of cells. When cells multiply in culture, the cell proliferation rate is sometimes measured by the amount of time the cells need to double their numbers (also called doubling times ).

The term “growing” or “growth” as used herein describes the process of increasing the number of pluripotent stem cells by culturing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 75%, 90%, 100%, 200%, 500%, 1000% or more, as well as the levels indicated in percentages. It should be understood that the number of pluripotent stem cells that can be obtained from a single pluripotent stem cell depends on the proliferation ability of pluripotent stem cells. The ability to proliferate pluripotent stem cells can be calculated from the cell doubling time, i.e. the time the cell needs to undergo mitotic division in culture, as well as the time during which pluripotent stem cells can be maintained in an undifferentiated state, which is equivalent to the number of passages multiplied by the number of days between each passage.

Differentiation is the process by which a unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell, such as a nerve cell or a muscle cell. A differentiated cell or cell induced by differentiation is a cell that has taken a more specialized (“committed”) position in the lineage of the cell. The term “committed” as applied to the differentiation process refers to a cell that has reached the stage of differentiation to a stage from which under normal conditions it will continue to differentiate to a particular cell type or a subset of cell types and will not be able to differentiate to a different cell type less differentiated type. “Dedifferentiation” refers to the process by which a cell returns to a less specialized (or committed) position in the cell lineage. As used herein, the term "cell lineage" refers to the heredity of the cell, i.e. from which cells the given cell originated and which cells it can give rise to. In the cell line of differentiation, the cell is placed in the hereditary scheme of development and differentiation. A differentiation line-specific marker refers to a characteristic specifically associated with the phenotype of cells of a differentiation line of interest, and can be used to assess the differentiation of a non-committed cell into cells of a differentiation line of interest.

In this document, the term "markers" means molecules of nucleic acids or polypeptides with differential expression in the studied cells. In this context, differential expression is understood as an increased level of a positive marker and a reduced level of a negative marker in comparison with an undifferentiated cell. The detectable level of a marker nucleic acid or polypeptide in the cells of interest is significantly higher or lower than other cells, which allows the cell of interest to be identified and distinguished from other cells using any of a variety of methods known in the art.

In this document, a cell is “positive for” a given marker or “positive” if the specified marker is clearly detected in the cell. Similarly, a cell is "negative for" a given marker or "negative" if the specified marker is not found in the cell. In particular, FACS positivity is usually higher than 2%, while the FACS negative limit is usually less than 1%. PCR positivity is typically less than 34 cycles (Cts); while PCR negativity is usually over 34.5 cycles.

In this document, the terms “cell density” and “seeding density” are used interchangeably and refer to the number of cells sown per unit surface area of a solid or semi-solid flat or curvilinear substrate.

In this document, the term "suspension culture" refers to a cell culture, individual cells or clusters suspended in a medium, and not attached to any surface.

In this document, the term "serum-free" refers to a medium devoid of human or animal serum. Accordingly, the serum-free culture medium does not contain serum or portions of serum.

In an attempt to reproduce the differentiation of pluripotent stem cells into functional pancreatic endocrine cells in a cell culture, the differentiation process is often seen as progression through several successive stages. In this document, the various stages are determined by the cultivation time and reagents described in the examples given here.

In this document, "definitive endoderm" refers to cells that have the characteristics of cells derived from epiblast during gastrulation and that form the gastrointestinal tract and its derivatives. Definitive endoderm cells express at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3-β (HNF3β)), GATA4, GATA6, MNX1, SOX17, CXCR4, Cerberus, OTX2, brachyury, goosecoid, C-Kit, CD99 and MIXL1. Marker characteristics of definitive endoderm cells include CXCR4, FOXA2 and SOX17. Thus, definitive endoderm cells can be characterized by their expression of CXCR4, FOXA2 and SOX17. Additionally, depending on the length of time during which cells are allowed to remain in stage 1, growth in HNF4α can be observed.

As used herein, the term “pancreatic endocrine cell” refers to cells capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide. In addition to these hormones, marker characteristics of pancreatic endocrine cells include one or more of NGN3, NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, and PAX6. Pancreatic endocrine cells expressing markers of beta cells may be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, MAFAPAX4 and PAX6.

In this document, the expressions “d1”, “d 1” and “day 1” are used interchangeably; "D2", "d 2" and "day 2"; "D3", "d 3" and "day 3" and so on. These combinations of numbers and letters refer to specific days of incubation at various stages in the process of stepwise differentiation protocol of this application.

The terms “glucose” and “D-glucose” are used interchangeably herein and refer to dextrose, a sugar widely found in nature.

In this document, the terms "NeuroD" and "NeuroD1" are used interchangeably to refer to the protein expressed in the progenitor cells of pancreatic endocrine cells, and the gene encoding it.

“LDN” and “LDN-193189” refers to ((6- (4- (2- (piperidin-1-yl) ethoxy) phenyl) -3- (pyridin-4-yl) pyrazolo [1,5-a] pyrimidine hydrochloride; DM-3189)) a BMP receptor inhibitor available under the trademark STEMOLECULE ^TM from Stemgent, Inc., Cambridge, Massachusetts, USA.

Isolation, reproduction and cultivation of pluripotent stem cells

Pluripotent stem cells can express one or more of the indicated TRA-1-60 and TRA-1-81 antibodies (Thomson et al. 1998, Science 282: 1145-1147). In vitro differentiation of pluripotent stem cells leads to loss of TRA-1-60 and TRA-1-81 expression. Undifferentiated pluripotent stem cells, as a rule, have alkaline phosphatase activity, which can be detected by treating the cells with 4% paraformaldehyde solution and then growing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Inc., Burrheim, California, USA). Undifferentiated pluripotent stem cells also tend to express OCT4 and TERT, as determined by RT-PCR.

Another desirable phenotypic property of grown pluripotent cells is the potential for differentiation into cells of all three germ layers: endodermal, mesodermal, and ectodermal tissues. Pluripotency of stem cells can be confirmed, for example, by injecting cells into mice with severe combined immunodeficiency (SCID), fixing the resulting teratomas with 4% paraformaldehyde and their subsequent histological examination for the presence of cell types derived from three germ layers. Pluripotency can alternatively be identified by creating embryoid bodies and analyzing embryoid bodies for the presence of markers associated with three germ layers.

Grown lines of pluripotent stem cells can be karyotyped using the standard G-banding method and compared with the published karyotypes of the respective primate species. It is desirable to obtain cells that have a “normal karyotype,” that is, euploid cells in which all human chromosomes are present and have no visible changes. Pluripotent cells can be easily propagated in culture by using different nutrient layers or by using vessels coated with matrix proteins. Alternatively, for standard cell multiplication, chemically defined surfaces can be used in combination with media of specific composition, such as mTeSR ^® 1 media (StemCell Technologies, Vancouver, British Columbia, Canada).

Cultivation in suspension culture in accordance with the method indicated in some embodiments of the present invention is carried out by planting pluripotent stem cells in a culture vessel, at a cell density that promotes cell survival and proliferation, but limits differentiation. As a rule, seeding density is applied, which ensures the absence of cell differentiation. It should be borne in mind that, although it is possible to sow suspensions of individual stem cells, it is preferable to sow small clumps of cells.

To provide pluripotent stem cells with sufficient and constant supply of nutrients and growth factors in suspension culture, the culture medium can be replaced or replenished on a daily basis or according to a predetermined schedule, for example, every 1-5 days. Large clusters of pluripotent stem cells can lead to the onset of cell differentiation, so measures can be taken to avoid the emergence of large aggregates of pluripotent stem cells. In accordance with some embodiments of the invention, the clusters of pluripotent stem cells dissociate, for example, every 2-7 days, and individual cells or small clusters of cells are either separated and transferred to another culture vessel (i.e. they are subcultured), or left in the same culture vessel and replace or replenish the culture medium.

Large clusters of pluripotent stem cells, including the precipitate of pluripotent stem cells obtained by centrifugation, can be subjected to enzymatic cleavage and / or mechanical dissociation. Enzymatic cleavage of clusters of pluripotent stem cells may be accomplished by placing the cell cluster in the enzyme, for example in type IV collagenase, Dispase ^® or Accutase ^®. Mechanical dissociation of large clusters of pluripotent stem cells can be performed using a device designed to separate clusters to a given size. Additionally or alternatively, mechanical dissociation can be performed manually with a needle or pipette.

A culture vessel for cultivating pluripotent stem cells in suspension in accordance with the methods described in some embodiments of the invention may be any tissue culture vessel (for example, having a sufficient level of purity for cultivating pluripotent stem cells), the inner surface of which is designed so that the pluripotent stem cells cultured in it cannot be held or attached to such a surface (for example, a vessel not treated for tissue tivirovaniya to prevent attachment or adhering to the surface). To obtain the required growing culture, cultivation in accordance with some embodiments of the invention is carried out using a controlled cultivation system (preferably a computer-controlled cultivation system), in which culture parameters such as temperature, agitation, pH and oxygen level are automatically controlled and regulated with using a suitable device. Once the desired culture parameters have been determined, the system can be configured to automatically adjust culture parameters as needed to improve growth and differentiation of pluripotent stem cells.

Pluripotent stem cells can be cultivated under dynamic conditions (i.e., under conditions in which pluripotent stem cells constantly move in suspension culture, for example, in a mixed suspension culture system) or in non-dynamic conditions (for example, in a static culture), preserving their ability proliferation, pluripotency, and karyotypic stability over several transfers.

For non-dynamic cultivation of pluripotent stem cells, pluripotent stem cells can be cultured in Petri dishes, T-flasks, HyperFlasks ^® (Corning Incorporated, Corning, NY, USA), CellStacks ^® (Corning Incorporated, Corning, New York, USA) or Cell Factories (NUNC ™ Cell Factory ™ Systems (Thermo Fisher Scientific, Inc., Pittsburgh, Pennsylvania, USA)) with or without coating. For dynamic cultivation of pluripotent stem cells, pluripotent stem cells can be cultured in a suitable vessel, for example, centrifuge tubes or Erlenmeyer flasks, on stainless steel, glass, in a disposable plastic shaker or in a blender vessel. The culture vessel can be connected to the control unit and, thus, constitute a controlled culture system. Mixing in the culture vessel (for example, a centrifuge tube or an Erlenmeyer flask) is carried out continuously or periodically. Mixing in the culture vessel is carried out to the extent that the pluripotent stem cells are maintained in suspension.

Pluripotent stem cells can be grown in any environment that provides enough nutrients and external stimuli to stimulate growth and division. Suitable media include E8 ™, IH3 and mTeSR ^® 1 or mTeSR ^® 2. The medium can be periodically replaced to renew nutrient supply and remove by-products secreted by cells. In accordance with some embodiments of the invention, the culture medium was changed daily.

SOURCES OF PLURIPOTENT STEM CELLS

Any pluripotent stem cells can be used in the methods of the present invention. Types of pluripotent stem cells that can be used include resistant lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as blastocyst), fetal tissue, or fetal tissue taken at any time during the gestation period as a rule, but not necessarily, up to a period of about 10-12 weeks of gestation. Non-limiting examples are resistant lines of human embryonic stem cells (hES) or human embryonic germ cells, such as, for example, human embryonic stem cells of H1, H7 and H9 lines (WiCell Research Institute, Madison, Wis., USA). Also acceptable are cells taken from a population of pluripotent stem cells already cultured in the absence of feeding cells.

Also consistent with the objectives of the present invention are inducible pluripotent cells (IPS) or reprogrammed pluripotent cells that can be obtained from adult somatic cells by forcibly expressing a number of factors related to pluripotent transcription factors such as OCT4, NANOG, Sox2, KLF4 and ZFP42 ( Annu Rev Genomics Hum Genet 2011, 12: 165-185). Human embryonic stem cells used in the methods of the present invention can also be prepared as described by Thomson et al. (US Patent No. 5,843,780; Science, 1998 , 282: 1145-1147; Curr Top Dev Biol 1998, 38: 133- 165; Proc Natl Acad Sci USA 1995, 92: 7844-7848). Also suitable are mutant lines of human embryonic stem cells, such as, for example, BG01v (BresaGen, Athens, Ga.), Or cells derived from mature human somatic cells, such as, for example, the cells described in Takahashi et al., Cell 131: 1-12 (2007). Pluripotent stem cells suitable for use in the present invention can be obtained using the methods described in Li and co-authors: ( Cell Cell Cell 4: 16-19, 2009); Maherali et al. ( Cell Stem Cell 1: 55-70, 2007); Stadtfeld et al. ( Cell Stem Cell 2: 230-240); Nakagawa et al. ( Nature Biotechnology 26: 101-106, 2008); Takahashi et al. ( Cell 131: 861-872, 2007); and publication of US Patent Application No. 2011-0104805. Other sources of pluripotent stem cells include induced pluripotent cells (IPS, Cell , 126 (4): 663-676). Other sources of cells suitable for use in the methods of the invention include cells derived from human umbilical cord tissue, cells derived from amniotic fluid, cells, cells derived from human placenta, and human parthenotes. In one embodiment, cells derived from human umbilical cord tissue may be obtained using the methods described in US Pat. No. 7,510,873, the disclosure of which is incorporated by reference in its entirety, where they relate to cell isolation and characterization. In another embodiment, cells derived from placental tissue can be obtained using the methods described in US Patent Application No. 2005/0058631, the disclosure of which is incorporated by reference in its entirety as it relates to the isolation and characterization of cells . In another embodiment, cells derived from amniotic fluid can be obtained using the methods described in US patent application US No. 2007/0122903, the disclosure of which is incorporated by reference in its entirety as it relates to the isolation and characterization of cells.

The characteristics of pluripotent stem cells are well known to those skilled in the art, and the identification of additional characteristics of pluripotent stem cells continues. Pluripotent stem cell markers include, for example, the expression of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the following markers: ABCG2 , cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. In one embodiment, pluripotent stem cells suitable for use in the methods of the invention express one or more (for example, 1, 2, 3 or all) markers CD9, SSEA4, TRA-1-60 and TRA-1-81 and are devoid of expression of the CXCR4 differentiation marker (also known as CD184), as determined by flow cytometry. In another embodiment, pluripotent stem cells suitable for use in the methods of the invention express one or more (for example, 1, 2 or all) markers of CD9, NANOG and POU5F1 / OCT4, as determined by RT-PCR.

Examples of pluripotent stem cells include the H9 human embryonic stem cell line (NIH code: WA09), H1 human embryonic stem cells (NIH: WA01 code), H7 human embryonic stem cells (NIH code: WA07), and human embryonic stem cells lines SA002 (Cellartis, Sweden). In one embodiment of the present invention, said pluripotent stem cells are embryonic stem cells, for example, H1 hES cells. In alternative embodiments, pluripotent stem cells of non-embryonic origin are used.

Differentiation of cells expressing markers characteristic of the pancreatic endoderm line from pluripotent stem cells

Growth of pluripotent stem cells

The present invention, in some embodiments, as described below, relates to the isolation and cultivation of stem cells, in particular the cultivation of stem cell clusters that retain pluripotency in a dynamic suspension culture system. Pluripotent cell clusters can be differentiated to produce functional β cells.

The pluripotent stem cells used in the methods of the present invention are preferably grown in a dynamic culture suspension prior to differentiation to the desired end point. It was found that pluripotent stem cells can be cultured and grow as clusters of cells in suspension in a suitable medium without loss of pluripotency. Such cultivation can occur in a dynamic suspension culture system where cells or cell clusters are kept in motion to prevent loss of pluripotency. Useful dynamic suspension culture systems include systems equipped with means for maintaining movement in culture, for example, by mixing, shaking, recirculating, or passing gases through a medium. Such mixing can be intermittent or continuous, it should provide sufficient movement of clusters of cells to promote growth and prevent premature differentiation. Preferably, the mixing comprises continuous mixing, for example, by means of a blade rotating at a certain speed. The blade may be rounded or flat. The speed of the blade should be such that the clusters are maintained in a suspended state and the subsidence is minimal. In addition, the blade angle can be changed to facilitate the upward movement of cells and cell clusters to avoid settling. In addition, the type of impeller, angle and speed of rotation can be coordinated in such a way that the cells and clusters will be maintained in a state of uniform colloidal suspension.

Suspension cultivation and cluster growth of pluripotent stem cells can be achieved by transferring static cultured stem cells to an appropriate dynamic culture system, such as disposable plastic, reusable plastic, made of stainless steel or glass vessels, such as a centrifuge tube or an Erlenmeyer flask. For example, stem cells cultured in an adherent static medium, i.e. on the surface of the carriers, can be first removed from the surface by the method of treatment with a chelating agent or enzyme. Suitable enzymes include, without limitation, the following: collagenase type ^{I, Dispase ® (Sigma Aldrich LLC} , St. Louis, Missouri, USA) or the commercially available formulation marketed under the tradename Accutase ^® (Sigma Aldrich LLC, of St. Louis, Missouri, USA). Accutase ^® is a cell detachment solution containing collagenolytic and proteolytic enzymes (isolated from crustaceans) and does not contain products isolated from mammals or bacteria. Thus, in one embodiment, the enzyme is a collagenolytic, or proteolytic enzyme, or cell detachment solution containing collagenolytic and proteolytic enzymes. Suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA). In some embodiments, the implementation of pluripotent stem cells of the culture is incubated with an enzyme or chelating agent, preferably until the edges of the colony begin to fold and separate, but until complete separation of the colonies from the surface of the culture. In one embodiment, cell cultures are incubated at room temperature. In one embodiment, the cells are incubated at a temperature above 20 ° C, above 25 ° C, above 30 ° C or above 35 ° C, for example, at a temperature between about 20 ° C and about 40 ° C, from about 25 ° C to about 40 ° C., from about 30 ° C. to about 40 ° C., for example, about 37 ° C. In one embodiment, the cells are incubated for at least about 1, at least about 5, at least about 10, at least about 15, at least about 20 minutes, for example, from about 1 to about 30 minutes, from about 5 to about 30 minutes, from about 10 to about 25 minutes, from 15 to 25 minutes, for example, about 20 minutes. In one embodiment, the method includes a step of removing the enzyme or chelating agent from the cell culture after treatment. In one embodiment, the cell culture is washed once or twice or more after removal of the enzyme or chelating agent. In one embodiment, the cell culture is washed with a suitable culture medium, such as mTeSR ^® 1 (Stem Cell Technologies, Vancouver, BC, Canada). In one embodiment, an Rho kinase inhibitor is used (eg, Y-27632, Axxora catalog number ALX-270-333, San Diego, CA). The Rho kinase inhibitor may be in a concentration of from about 1 to about 100 μM, about 1 to 90 μM, from about 1 to about 80 μM, from about 1 to about 70 μM, from about 1 to about 60 μM, from about 1 to about 50 μmol, from about 1 to about 40 μmol, from about 1 to about 30 μmol, from about 1 to about 20 μmol, from about 1 to about 15 μmol, from about 1 to about 10 μmol or 10 μmol. In one embodiment, the Rho kinase inhibitor is added in an amount of at least 1 μM, at least 5 μM, or at least 10 μM. Cells can be removed from the surface of a static cultivation system with a scraper or rubber rod. The medium and cells can be transferred to a dynamic culture system using a glass pipette or other suitable means. In a preferred embodiment, the medium in the dynamic culture system is replaced daily.

The invention provides, in one embodiment, methods for cultivating and growing pluripotent stem cells in a three-dimensional suspension culture. In particular, methods are provided for the cultivation and growth of pluripotent stem cells by forming aggregated clusters of cells of these pluripotent stem cells. Cell clusters may form by treating the pluripotent stem cell cultures enzyme (e.g., a neutral protease, or Dispase ^®) or chelating agent prior to culturing the cells. Preferably, the cells are cultured in a stirred or shaken suspension culture system. In one embodiment of the present invention, the formation from these clusters of pluripotent stem cells of differentiated cells expressing markers characteristic of the pancreatic endoderm cell line is also presented.

Preferably, cell clusters consist of aggregated pluripotent stem cells. Aggregated stem cells express one or more markers of pluripotency, for example, one or more (for example, 1, 2, 3 or all) of the markers CD9, SSEA4, TRA-1-60, TRA- and 1-81, and also show the absence of expression one or more differentiation markers, for example, are deprived of CXCR4 expression. In one embodiment, the aggregated stem cells express the pluripotency markers CD9, SSEA4, TRA-1-60, TRA-and 1-81, and lack expression of the CXCR4 differentiation marker

One embodiment is a method for cultivating pluripotent stem cells as cell clusters in suspension culture. Cell clusters are aggregated pluripotent stem cells cultured in a dynamic stirred or shaken suspension culture system. Cell clusters can be transferred from a flat attached culture by adding an enzyme, such as a neutral protease, such as Dispase, as a cell-separating agent to a stirred or shaken suspension culture system. Examples of suitable enzymes include, without limitation, type IV collagenase, Dispase ^® or Accutase ^®. Cells maintain pluripotency in a stirred or shaken culture system, in particular in a stirred suspension culture system.

Another embodiment of the invention relates to a method of cultivating pluripotent stem cells as cell clusters in suspension culture, in which clusters of cells are aggregated pluripotent stem cells displaced from a flat attached culture using a chelating agent, for example, EDTA, and cultured in a stirred or shaken suspension culture system. Cell clusters support pluripotency in a stirred or shaken suspension culture system, in particular in a mixed suspension culture system.

Another embodiment of the invention relates to a method of cultivating pluripotent stem cells as cell clusters in suspension culture, in which the cell clusters are aggregated pluripotent stem cells transferred from a flat attached culture using the enzyme Accutase ^® and cultured in a stirred or shaken suspension culture system. Cell clusters support pluripotency in a dynamic suspension culture system.

The cell clusters of the invention can be differentiated into mesodermal cells, such as heart cells, ectoderm cells, nerve cells, single-hormone positive cells, or pancreatic endoderm cells. The method may additionally include differentiation, for example, differentiation of pancreatic endoderm cells into progenitor cells of pancreatic cells and cells expressing pancreatic hormone. In another embodiment, the progenitor cells of pancreatic cells are characterized by the expression of the transcription factors β-cells Pdx1 and Nkx6.1.

In one embodiment, the stage of differentiation takes place after at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours at least 144 hours, at least 168 hours, at least 196 hours, or, more preferably, from about 48 hours to about 72 hours in a suspension culture system. Differentiation can be carried out using stepwise progression of environmental components, for example, as described in the examples (for example, see Table A and Tables 1a and 1c).

In a preferred embodiment, a three-dimensional cluster of cells is obtained by growing pluripotent stem cells in a flat, attached culture; growing pluripotent stem cells into aggregated cell clusters; and transferring clusters of pluripotent stem cells from an attached flat culture to a dynamic culture suspension using an enzyme or chelating agent. Another preferred embodiment is a method of growing and differentiating pluripotent stem cells in a dynamically mixed suspension culture system by growing pluripotent stem cells in a flat, attached culture; growing pluripotent stem cells into aggregated cell clusters; and transferring clusters of pluripotent stem cells from an attached flat culture to a dynamic culture suspension using an enzyme or chelating agent; and differentiation of clusters of pluripotent cells in a dynamic stirred suspension culture system to create a population of pancreatic progenitor cells.

Another embodiment is an isolated stem cell transplant product containing differentiated stem cells obtained from a suspension of expanded clusters of pluripotent stem cells that differentiate into progenitor cells of pancreatic cells. More specifically, the isolated stem cell transplant product is obtained by growing pluripotent stem cells in a flat attached culture; growing pluripotent stem cells into aggregated cell clusters; and transferring clusters of pluripotent stem cells from an attached flat culture to a dynamic culture suspension using an enzyme or chelating agent; and differentiation of clusters of pluripotent cells in a dynamically stirred suspension culture system. The isolated stem cell transplant product is preferably used for the treatment of diabetes.

In another embodiment, the method includes transplantation into an animal suffering from diabetes for further maturation in vivo into functional pancreatic endocrine cells.

Another embodiment is a method of growing and differentiating pluripotent stem cells in a suspension culture system containing growing pluripotent stem cells in a flat, attached culture; removing pluripotent stem cells from a flat attached culture using an enzyme; attaching pluripotent stem cells to microcarriers in static culture; growing pluripotent cells in a dynamically stirred suspension culture system; and differentiation of pluripotent cells in a dynamically mixed suspension culture system to create a population of pancreatic progenitor cells.

Microcarriers can be of any form known in the art of cell attachment, in particular, microcarriers can be spheres. The microcarrier may consist of materials of natural or synthetic origin. Examples include collagen based microcarriers, dextran-based or cellulose-based. For example, the microcarrier spheres can be modified polystyrene spheres with cationic trimethyl ammonium attached to the surface to provide the microcarrier with a positively charged surface. The diameter of the sphere can range from about 90 to about 200 microns, alternatively from about 100 to about 190 microns, alternatively from about 110 to about 180 microns, in an alternative embodiment, from about 125 to 175 microns in diameter. The spheres of microcarriers may consist of a thin layer of denatured collagen, chemically bound to a matrix of cross-linked dextran. Microcarrier spheres can consist of the following materials: glass, ceramics, polymers (such as polystyrene), or metals. In addition, microcarriers can be uncoated or coated, for example, of silicon or protein, such as collagen. In an additional aspect, the microcarrier may consist of or have a coating of compounds that improve the binding of the cell to the microcarrier and improve the separation of the cell from the microcarrier, including, among other things, compounds such as sodium hyaluronate, poly (monostearoylglyceride-co-succinic acid), poly-D, L-lactide-co-glycolide, fibronectin, laminin, elastin, lysine, n-isopropylacrylamide, vitronectin and collagen. Examples further include microcarriers having microcurrents, such as microcarriers with a partial galvanic pair of zinc and copper, which produces low levels of biologically significant electricity; or microcarriers that are paramagnetic, such as paramagnetic calcium-alginate microcarriers.

In some embodiments, a population of pancreatic endoderm cells is produced by phased differentiation of pluripotent cell clusters. In some embodiments, the implementation of pluripotent cells are human embryonic pluripotent stem cells. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a precursor cell of a primary strip. In an alternative aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a mesentoderm cell.

In some embodiments, the implementation of the present invention relates to a method of phased differentiation of pluripotent cells, containing the cultivation of cells in stages 3-5 in a dynamic suspension culture. In some embodiments, the formed pancreatic endodermal population is transplanted into a diabetic animal for further maturation in vivo to functional endocrine cells. The present invention also provides systems or kits for use with the methods of the present invention.

The present invention also represents a cell or population of cells that can be obtained by the methods of the present invention. The present invention also provides a cell or cell population obtained by the methods of the present invention.

The invention provides processing methods. In particular, the present invention provides a method for treating a patient suffering from diabetes or at risk of developing the disease.

The present invention also provides a cell or population of cells that can be obtained or obtained by the method of the present invention for use in a processing method. In particular, the present invention also represents a cell or a population of cells that can be obtained or obtained by the method of the present invention for use in treating a patient with diabetes or at risk of developing the disease. Diabetes can be type I or type II diabetes.

In one of the embodiments, the processing method includes the implantation of cells that are obtained or can be obtained by the method of the present invention, the patient.

In one embodiment, the processing method comprises differentiating pluripotent stem cells in a test tube in stage 1, stage 2, stage 3, stage 4 or stage 5 cells, for example, as described herein, and implanting differentiated cells into the patient's body.

In one embodiment, the method also comprises the step of cultivating pluripotent stem cells, for example, as described herein, prior to the differentiation step of pluripotent stem cells.

In one embodiment, the method also includes a step of differentiating cells in vivo, after an implantation step.

In one embodiment, the patient is a mammal, preferably a human.

In one embodiment, the implementation of the cells can be implanted in the form of dispersed cells or cells forming clusters, which can be entered into the portal vein of the liver by the method of infusion. Cells can alternatively be introduced into biocompatible, degradable polymeric support materials, porous, non-degradable devices or in encapsulated form to protect against the immune response of the host organism. Cells can be implanted in a suitable place in the body of the recipient. Implantation sites include, for example, the liver, the natural pancreas, the space below the renal capsule, the omentum, the abdominal cavity, the subserous space, the intestine, the stomach, or the subcutaneous pocket.

To stimulate further differentiation, survival or activity of the implanted cells in vivo, before, simultaneously with or after the introduction of the cells, additional factors can be introduced, such as growth factors, antioxidants or anti-inflammatory substances. These factors can be secreted by endogenous cells and act on the embedded cells in situ. Differentiation of implanted cells can be induced by any combination of endogenous growth factors known in the art and exogenous administration of the growth factors known in the art.

The number of cells used in implantation depends on a number of different factors, including the condition of the patient and his response to treatment, and can be determined by a person skilled in the art.

In one embodiment, the processing method also includes embedding the cells prior to implantation into a three-dimensional support material. Cells can be maintained in vitro on this support material prior to implantation into a patient. Alternatively, the supporting material containing cells can be implanted directly into the patient's body without additional in vitro culture. The supporting material may optionally include at least one pharmaceutical substance that ensures the survival and functioning of the transplanted cells.

In some embodiments, implementation of the present invention, one or more of the following options can be used in the methods of the present invention.

Table A Components / Terms Stage Suitable quantities Activin A (AA) 1,3 Stage 1: approximately 100 mg / ml
Stage 3: about 5 ng / ml, from about 3 ng / ml to about 6 ng / ml AlbuMAX ^® 3-5 Approximately 0.1% ALK5 inhibitor 4, 5 About 1 μMol, about 500 to about 1000 nM, about 600 to about 1000 nM, about 700 to about 1000 nM, about 800 to about 1000 nM, about 100 nM, about 500 nM or about 1 μM, from about 0.6 to about 1 μm BSA 1-5 Approximately 2%, from 0.1% to approximately 2% Cypi (Cyp26 inhibitor) 4, 5 Approximately 100 nM, from about 80 nM to about 120 nM, from about 50 nM to about 150 nM FGF7 (“F7”) 2, 3 Approximately 50 ng / ml, from approximately 30 ng / ml to approximately 60 ng / ml, from approximately 25 ng / ml to approximately 55 ng / ml GDF8 one Approximately 100 ng / ml, from approximately 80 ng / ml to approximately 150 ng / ml, from approximately 75 ng / ml to approximately 125 ng / ml, from approximately 75 ng / ml to approximately 150 ng / ml Glucose 1-5 Stages 1-4:
Approximately 8 mmol, from about 1 mmol to about 8 mmol, from about 3 mmol to about 5 mmol
or
Stages 3-4:
Approximately 25 mmol, from about 10 to about 25 mmol
or
Stage 5
Less than about 11 mM, from about 1 mM to about 10 mM
or
Stage 5
More than about 25 mmol, from about 25 mmol to about 50 mmol ITS-X 1-5 Approximately 1:50 000, approximately 1: 200, approximately 1: 1000, approximately 1:10 000 LDN 3 Approximately 100 nM, from about 80 nM to about 120 nM, from about 50 nM to about 150 nM L-glutamine 1-5 Approximately 2 mM, from about 1 mM to about 3 mM, from about 2 mM to about 6 mM, from about 1 mM to about 6 mM Amount of lipids From approximately 0.1% to approximately 0.2%, from approximately 0.05% to approximately 0.15%, from approximately 0.15% to approximately 0.2% MCX one Approximately 3 μM, approximately 2 μM, from about 1 μM to about 5 μM, from about 2 μM to about 4 μM, from about 1 μM to about 3 μM, from about 2 μM to about 3 μM Amount of oxygen 1-5 Hypoxia to about 30% standard, from about 10% to about 25% standard, from about 15% to about 30% standard Retinoic acid 3 Approximately 2 μmol, from approximately 1 μmol to approximately 3 μmol, from approximately 1.5 μmol to approximately 2.5 μmol SANT 3, 4 Approximately 0.25 μmol, from about 0.1 μmol to about 0.3 μmol, from about 0.2 to about 0.3 μmol. from about 0.1 μmol to about 0.25 μmol SCIO (Alk5 inhibitor) four Approximately 100 nM, approximately 2 μM Time of differentiation of cells from pluripotent to definitive endoderm cells Less than 48 hours, less than 30 hours, less than 24 hours, less than 18 hours, approximately 18 to 30 hours TppB or TPB four Approximately 500 nM, approximately 100 nM, from about 50 nM to about 550 nM, from about 50 nM to about 150 nM, from about 200 nM to about 500 nM, from about 300 nM to about 550 nM, about 50 nM, from about 25 nm to about 75nM Wnt3A one Approximately 20 ng / ml, from approximately 10 ng / ml to approximately 25 ng / ml, from approximately 18 ng / ml to approximately 30 ng / ml, from approximately 18 ng / ml to approximately 22 ng / ml Y-27632 0 Approximately 10 μmol, from about 5 μmol to about 15 μmol, from about 5 μmol to about 10 μmol

Publications cited in this document are fully incorporated herein by reference. The present invention is further illustrated, inter alia, by the following examples.

EXAMPLES

THE PRESENT INVENTION IS FURTHER ILLUMINATED BY THE FOLLOWING UNLIMITED EXAMPLES.

EXAMPLE 1

SUSPENDING AND CLUSTERING OF HUMAN EMBRYONIC STEM CELLS OF CELL LINE H1 USING DISPASE / NEUTRAL PROTEASE

Cell line of human embryonic stem cells H1, (WA01 cells, WiCell, Madison, Wisconsin, USA) with passaging 41 times with PBS (№ catalog 14190, Invitrogen) and treated with 1 mg / ml Dispase ^® (Neutral Protease, Sigma Aldrich Co LLC, Catalog No. D4818, St. Louis, Missouri, USA) at DMEM / F12 (Invitrogen Catalog No. 11330, Grand Island, New York, USA). Cells were incubated at 37 ° C for 15-25 minutes, until the edges of the colony began to curl and move away, but before complete separation of the colonies from the surface of the culture. Dispase ^{® was} then removed, and the Petri dish was washed twice with mTeSR ^® 1 (Stem Cell Technologies, Vancouver, British Columbia, Canada) containing 10 μM Y-27632 (Axxora, catalog number ALX-270-333, San Diego, California, USA). The mTeSR ^® 1 medium containing 10 μM Y-27632 was then added to the culture dish in a quantity of 5 ml / 60 cm ² , and the cells were removed from the surface with a scraper or a rubber rod. The medium and cells were transferred to a 50 ml conical tube using a glass pipette and the clusters were centrifuged at 90 g (OCS, relative centrifugal force) for 3 minutes.

After centrifuging, the medium was aspirated and the cells were carefully resuspended and quickly crushed in 12 ml of mTeSR ^® 1 medium containing 10 μM Y-27632 for every 225-240 cm ^{2 of} total flat culture (equivalent to one T225 flask or four plates 10 cm, approximately 90 million cells). The cell suspension was then transferred (1 ml / well) to 6-well Ultra Low Binding Culture plates (Corning Incorporated, Cat. No. 3471, Corning, New York, USA) containing 2 ml / well of fresh mTeSR ^® 1 medium with 10 μM Y-27632. The cells thus separated resembled monolayer fragments with an average diameter of the lifted fragments of about 20–30 μmol (FIG. 1a), each of which consists of a cluster of cells. These monolayer fragments were incubated in suspension for 2 hours (the incubation time can be in the range from 0.5 to 4 hours), while dotted aggregates of fragments were observed. The aggregates are then quickly triturated with a glass pipette 10 ml of medium and incubated overnight (the aggregates can go directly into suspension) into a loose binding plate (the aggregates can also be incubated on untreated plastics for cell culture and processed plastics for cell culture).

After incubation overnight (18-24 hours), the cells and medium were transferred directly to a 125 ml centrifuge tube (Corning Incorporated, catalog number 4500-125, Corning, New York, USA) containing 25 ml of medium mTeSR ^® 1 and mixed at a speed of 50 revolutions per minute (range can be 30-80 + revolutions per minute) to obtain a final volume of approximately 75 ml. The medium was changed daily for 4 days. Pluripotency was determined after 4 days in culture and flow cytometry results showed high expression of pluripotency markers (CD9, SSEA4, TRA-1-60, TRA- and 1-81) with almost no expression of the differentiation marker (CXCR4). See fig. 1b. These data show that H1 HES cells can be successfully transferred as clusters of cells of a flat, attached culture into a suspension culture using Dispase ^® as a cell-separating agent and maintain pluripotency in a dynamic suspension culture system. This example can also be carried out by shaking, rather than by mixing the suspension system on media and in Erlenmeyer flasks with comparable results.

After 4 days in suspension culture (differentiation can also begin 24-120 hours after the formation of aggregates, preferably a culture should form 2-3 days before the start of differentiation), pluripotent cell aggregates are differentiated using stepwise progression of environmental components to induce cells to form pancreatic development. Centrifugal mixing to differentiate the aggregates takes place at a speed of 65 revolutions per minute. The medium and components are shown in Table 1a.

At the end of stage 1, the samples were subjected to flow cytometry and PCR. Suspension differentiated cultures formed a homogeneous and homogeneous cell population in the form of loose aggregates at the end of stage 1 (Fig. 1c), with practically no expression of the pluripotency marker (CD9), while the markers of the final differentiation of endoderm were quite high, 97.2% CXCR4 (CD184) -positive and 97.3% CD99-positive (fig.1D). These results correlate with the results of Qrt-PCR, which showed a sharp decrease in the expression of pluripotency genes (CD9, NANOG and POU5F1 / OCT4) and a large increase in the genes associated with definitive endoderm (CXCR4, Cerberus, GSC, Foxa2, GATA4, GATA6, MNX1 and SOX17) compared with undifferentiated cells of the HES WA01 line (Figure 1e).

The final endoderm clusters were then further differentiated towards the primitive gut by removing the substances of the TGF-β family, GDF8, and adding FGF7 to the medium. After three days of cultivation with FGF7, the clusters differentiated into pancreatic PDX1-expressing cells by adding all-trans-retinoic acid or to medium containing high glucose (25 mM) and a low concentration of lipid-rich bovine serum albumin (AlbuMAX ^® (Life Technologies Corporation, Carlsbad, California, USA)), or to a medium containing a relatively low concentration of glucose (8 mmol) and 2% bovine serum albumin without fatty acids. The detailed addition of components in these environments is listed in Table 1a. At the end of differentiation, samples were analyzed for the expression of pancreatic progenitor cell markers. It was noted that clusters differentiated with either low glucose + 2% FAF-BSA (A), or high glucose + 0.1% AlbuMAX ^® (B) - as determined by flow cytometry, expressed high levels NKX6.1, a necessary transcription factor for functional β-cells, and high levels of endocrine pancreatic markers, such as synaptophysin and chromogranin (Table 1b). These results are consistent with the results of RT-PCR, which showed high levels of several pancreatic progenitor genes expressed in samples taken on conditions A and B (data not shown).

Typical morphologies of cell clusters during progression through differentiation from definitive endoderm (DE) (Fig. 1c) to primitive gut cells and pancreatic endoderm (Fig. 1f) showed visible morphological changes in cells and cell clusters. As a rule, pluripotent clusters are dense and dark according to phase-contrast microscopy, then become less dense as the cells progress to primitive intestine cells in stage 2. This morphology reverses with subsequent trans-retinoic acid during subsequent processing and the clusters again become more dense and uniform, with a smooth cluster boundary.

Cells differentiated according to condition B in stage 4 were treated for an additional 5 days in stage 5 with medium containing the ALK5 inhibitor (table 1c). This period of additional maturation in culture leads to a significant increase in the expression of endocrine markers: INS, GCG, SST, PPY and PCSK1. Cell clusters were then implanted into the kidney capsule of SCID-BG mice in accordance with the approved IACUC test protocol, and then in mice for 20 weeks, the C-peptide level was measured every 2-4 weeks in a hungry and fed state. 4 weeks after implantation, after a 20-hour fast, and then stimulation with glucose, C-peptide was not detected. By week 6, 2 out of 5 mice showed some positive (0.087 and 0.137 ng / ml) amounts of human C-peptide, and by week 10, 5 out of 5 mice were positive (0.085 - 0.291 ng / ml) for C-peptide. After 16 weeks, after a 20-hour fast and stimulation with glucose, all 4 mice (4/4) showed positive (0.377–3.627 ng / ml) C-peptide expression.

These results indicate that aggregates of pluripotent cells can be formed and then differentiated in suspension culture to generate a population of pancreatic progenitor cells characterized by the expression of β-cell transcription factors, such as Pdx1 and Nkx6.1. Thus, differentiated cell clusters that are implanted and mature in vivo can express insulin in response to glucose load at physiologically relevant levels.

Table 1a
Differentiation protocol Stage 1 Stage 2 Stage 3 Stage 4 Base environment
(final glucose concentration) MCDB131
8mM glucose MCDB131
8 mmol (A) or
25 mM glucose (B) Protein supplements 2% fatty acid free bovine serum albumin (FAF-BSA) and 2 mM L-Glutamine 2% fatty acid free bovine serum albumin (FAF-BSA) and 2 mM L-Glutamine (A)
or 0.1% Albumax (bovine serum albumin) and 2 mM L-Glutamine (B) Growth factors
AND / OR
Small molecules MCX (3 μM)
0-24 hours
GDF8 (100 ng / ml) for 0-96 hours
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1: 200)
RA (2µmol)
SANT (0.25 μM)
AA (5 ng / ml)
LDN (100 nM) ITS-X (1: 200)
SANT (0.25 μM)
Cypi (100 nM)
TppB (500 nM)
LDN (100 nM) Total day four 3 four five Medium change Every 24 hours Every 24 hours Every 24 hours Every 24 hours

Table 1b
Flow cytometry results for selected differentiation markers Glucose (sample) BSA %
NKX6.1 %
CDX2 %
Insulin %
Synaptophysin %
Chromogranin Low (A) 2 $ FAF
BSA 55 7 5.5 34.5 26.5 High level (B) 0.1%
Albumax 48 7 0.5 26.9 thirty

Table 1c
Differentiation protocol Stage 5 Base environment
(final glucose concentration) MCDB131
(25mM glucose) Protein supplements 0.1% AlbuMAX ^® (bovine serum albumin)
and 2mM L-glutamine Growth factors
AND / OR
Small molecules ITS-X (1: 200)
Cypi (100 nM)
LDN (100 nM)
ALKVi (10mM) Total day five Medium change Every 24 hours

EXAMPLE 2

SUSPENDING AND CLUSTERING OF HUMAN EMBRYONIC STEM CELLS OF CELL LINE H1 BY EDTA

Human embryonic stem cell lines H1, (WA01, WiCell cells, Madison, WI, USA), when passaged, were washed once with PBS (Catalog No. 14190, Invitrogen) 41 times and treated with EDTA, a non-enzymatic cell-separating agent / passaging agent (Lonza , Catalog number 17-7-11 e). Cells were incubated at room temperature for 8 minutes. EDTA was then removed, and after 1 or 2 minutes (9-10 minutes of total EDTA exposure), the plate was washed with mTeSR ^® 1 containing 10 μM Y-27632 (Axxora No. ALX-270-333, San Diego, state California, USA) and separated cells were collected in a 50 ml conical tube using a glass pipette. After one additional washing cycle of the plate with mTeSR ^® 1 containing 10 μM Y-27632, the washings were collected and combined with the separated cells. It should be noted that some cells remained on the plate 9-10 minutes after contact with EDTA at room temperature, and the separated cells were not completely crushed into a suspension of individual cells. Instead, cells were removed from the surface in the form of small aggregates. Then the medium and the cells were transferred to a 50 ml conical tube using a glass pipette and cells were counted (NucleoCounter ^® -ChemoMetec A / S, catalog number YC-T100, Denmark). If necessary, additional mTeSR ^® 1 medium containing 10 μM Y-27632 was added in order to bring the cell concentration to 1.0-1.5 million cells / ml.

The cells were not centrifuged, since the clusters are freely aggregated and can divide into individual cells by centrifuging the pellet and resuspending it with a pipette. Instead, the medium and the cells in the tubes were gently mixed until a homogeneous suspension was formed. If desired, it is also possible to lengthen the EDTA treatment period and separate cells near a suspension of individual cells. The cell suspension was then transferred to 6-well plates treated with two non-woven cultures (Becton Dickinson, catalog number Falcon 351146, Franklin Lakes, NJ, USA) at 37 ° C in a humidified incubator with 5% CO ₂ , according to 3 ml / well from a glass pipette. Cells were incubated in suspension for 2 h, after which point aggregates were observed. The aggregates were then crushed by careful pipetting with a glass pipette to separate large aggregates and create a suspension of homogeneous, uniform clusters, then incubated overnight without exposure.

After 18-24 hours, cells and media were centrifuged in 50 ml of conical tubes at 90g (OCS) for 3 minutes. The spent medium supernatant was disposed of, the cell aggregates suspended in fresh mTeSR ^® 1, and the suspension was transferred to a centrifuge tube (Corning Incorporated, catalog number 4500-125, Corning, New York, USA), stirred at 55 revolutions per minute at 37 ° C in a humidified 5% CO ₂ incubator. The medium was changed daily for 2 days. Pluripotency was determined after 2 days in a mixed suspension of the culture before proceeding to differentiation of the culture. The results of flow cytometry for CD9, SSEA4, TRA-1-60, TRA-1-81 and CXCR4 expression are shown as a scatter plot in FIG. 2a These data show high expression of pluripotency markers (CD9, SSEA4, TRA-1-60, TRA-1-81) and low or absent expression of the differentiation marker (CXCR4). These results show that H1 HES cells can be transferred to a suspension culture from a flat, attached culture using a non-enzymatic method of separation and will maintain pluripotency in a dynamic suspension culture system.

After 2 days in suspension culture, pluripotent cell aggregates differentiate with a stepwise progression of environmental components to induce cells to form a pancreatic pathway. The mixing counter is maintained at a speed of 55 revolutions per minute. The medium and components are shown in Table 2a.

At the end of stage 1, the samples were subjected to flow cytometry and PCR. Suspension differentiated cultures formed a homogeneous and homogeneous cell population in the form of loose aggregates at the end of stage 1 (Fig.2b), with virtually no expression of the pluripotency marker (CD9), while expression of CXCR4 (CD184), a marker for differentiation of definitive endoderm, was sufficient high, 95.9% ± 1.8 sd (Fig. 2c) after three runs of the centrifuge. These results correlate with the results of Qrt-PCR, which showed a sharp decrease in the expression of pluripotency genes (CD9, NANOG and POU5F1 / OCT4) and a large increase in the genes associated with definitive endoderm (CXCR4, Cerberus, GSC, Foxa2, GATA4, GATA6, MNX1 and SOX17) compared with undifferentiated cells of the HES WA01 line (Fig. 2d).

Clusters of definitive endoderm from centrifuge tubes are then combined, transferred to either another centrifuge tube or an Erlenmeyer flask (with shaking system) and sent for further differentiation into primitive intestinal cells by removing GGF7 from the medium and adding FGF7. After three days of culture with FGF7, the clusters differentiated into pancreatic Pdx1-expressing cells by adding all-trans-retinoic acid to medium containing relatively low glucose concentrations (8 mM) and 2% bovine serum albumin free fatty acid. The detailed addition of components in these environments is listed in Table 2a. At the end of differentiation, samples were analyzed for the expression of pancreatic progenitor cell markers. As a result of flow cytometry, high levels of Nkx6.1, a transcription factor necessary for functional β-cells, and high levels of endocrine pancreatic markers, such as synaptophysin and chromogranin, were observed in both suspensions (Table 2b and Figure 2e). These results are consistent with the results of RT-PCR, which showed very similar high levels of several pancreatic progenitor genes expressed in samples obtained in centrifuge tubes or Erlenmeyer flasks (Fig. 2F).

These results show that pluripotent cell aggregates can be formed and then differentiated in suspension culture in different suspension culture formats, including a stirred or shake suspension system to generate a population of pancreatic progenitor cells characterized by the expression of β-cell transcription factors, such as Pdx1 and Nkx6.1.

Table 2a
Environment components and differentiation protocol Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Base environment mTeSR ^® 1 MCDB131
(8mM glucose)
3.64 g / l NaCO ₃ MCDB131
(8mM glucose)
2.41 g / l NaCO ₃ Supplements mTeSR ^® 1 2% FAF-BSA
1:50 000 ITS-X
1x GlutaMax 2% FAF-BSA
1: 200 ITS-X
1x GlutaMax Growth factors GDF8 (d2 only)
100 ng / ml FGF7
50 ng / ml FGF7
50 ng / ml Small molecules Y-27632
( day 1 only )
[10 μm] MCX
(0-24 hours)
[2 μm] RA [2 μM]
SANT [0.25 μM]
TPPB [100 nM]
LDN (day 1 only) [100 nM] SANT [0.25 μM]
Cypi [100 nM]
ALK5 inhibitor [1 μM]
TPPB [100 nM] Day 3 3 3 3 3 NOTES : 1 with NTCT
2 days SF Medium change
Day 1 and 2,
No replacement for day 3 Medium change
Day 1 and 3,
No replacement for day 2 Medium change
Day 1 and 2,
No replacement for day 3 Replacing the environment on day 1
and day 2,
No replacement for day 3

Table 2b
Flow cytometry results for selected differentiation markers Sample %
NKX6.1 %
CDX2 %
SOX2 %
NKX2.2 %
Synaptophysin %
Chromogranin Centrifuge Tubes (avg.) 68.1 4.0 31.3 36.2 27.0 25.3 Erlenmeyer flask 65,8 7.9 28.1 30.0 30.7 17.0

EXAMPLE 3

SUSPENSION CLUSTERING AND SERIAL SUSPENSION PASSAGE OF HUMAN EMBRYONIC STEM CELLS OF H1 CELL CELL LINE

Cell line of human embryonic stem cells H1, (WA01 cells, WiCell, Madison, Wisconsin, USA) in 40 passaging grown on tissue culture treated polystyrene coated with Matrigel ^® (Corning Incorporated, Corning, NY, USA), washed twice with PBS (catalog number 14190, Invitrogen) and treated with a solution of half Accutase ^® efficiency (one part to one part PBS Accutase ^®, Sigma-Aldrich, № catalog a-6964, St. Louis, Missouri , USA). Cells were incubated at room temperature for 3 and a half minutes. (Accutase ^® is a cell detachment solution consisting of collagenolytic and proteolytic enzymes (isolated from crustaceans) and not containing products isolated from mammals or bacteria). Then Accutase ^® was removed and after 3 minutes (6 minutes total exposure to 1/2 Accutase ^®) plate was washed mTeSR ^® 1 medium containing 10 micromolar Y-27632 and the separated cells were collected into 50 ml conical tube using a glass pipette. After one additional washing cycle of the plate with mTeSR ^® 1 containing 10 μM Y-27632, the washings were collected and combined with the separated cells. Some cells remained on the plate after exposure to Accutase ^® and the separated cells were not completely ground into a single cell suspension. Cells were removed from the surface in the form of small aggregates (Fig. 3A). The medium and cells were transferred to a 50 ml conical tube using a glass pipette and cells were counted. If necessary, additional mTeSR®1 medium containing 10 μM Y-27632 was added in order to bring the cell concentration to 1.0-1.5 million cells / ml.

The cells were not centrifuged, since the clusters are freely aggregated and can divide into individual cells by centrifuging the pellet and resuspending it with a pipette. Instead, the medium and the cells in the tubes were gently mixed until a homogeneous suspension was formed. Then, the cell suspension was transferred to a 6-well Ultra Low Binding Culture plate at 37 ° C in a humidified 5% CO2 incubator, 3 ml / well, using a glass pipette. Cells were incubated in suspension for 90 minutes, after which point aggregates were observed. The aggregates were then briefly processed and transferred directly to a 125 ml centrifuge tube containing 25 ml of mTeSR®1 medium mixed at 55 revolutions per minute (the final volume was about 75 ml). The medium is changed daily for 3 days, and pluripotency in culture was determined for 3 days. Cluster images obtained using phase-contrast microscopy showed a population of spherical clusters that formed after 90 minutes in a static suspension culture after growing for three days in culture (FIG. 3b). At the end of three days in suspension culture, cells are analyzed for pluripotency by flow cytometry for the presence of CD9, SSEA4, TRA-1-60, TRA-1-81 and CXCR4 markers. Cells maintained a high level of expression of pluripotency markers (CD9, SSEA4, TRA-1-60, TRA-1-81), while there was practically no expression of CXCR4, a differentiation marker (Table 3). These results show that H1 hES cell line may be transferred to suspension culture of a flat attached culture using the enzymatic separation method, for example using Accutase ^®, and maintain pluripotency in dynamic suspension culture system.

The pluripotent clusters were then sequentially passaged using Accutase ^® dissociation for an additional 20 passages. At each passage, 50 million cells were precipitated by gravity for 2 minutes in a 50 ml conical tube, washed twice with PBS and treated with a half-efficiency Accutase ^® solution in a water bath at 37 ° C with gentle rotation of the tube at the second and fourth minute after adding Accutase ^® . After six minutes of incubation, Accutase ^® was removed from the tube without disturbing the cell pellet. The cells were then incubated for a further 3 minutes (9 minutes total exposure Accutase ^®). The tube was then washed with mTeSR®1 containing 10 μM Y-27632, crushed twice with a glass pipette, and the suspended cells were passed through a 70 μM cell filter (BD Falcon, Cat. No. 352350, Franklin Lakes, New Jersey , USA). Two additional washes were performed with the mTeSR®1 medium containing 10 μM Y-27632 and passed through a cell filter.

The medium and cells in the tube were gently mixed until a homogeneous suspension was formed. Then the cell suspension was transferred to a 6-well Ultra Low Binding Culture plate at 37 ° C in a 3 ml / well humidified 5% CO2 incubator using a glass pipette and incubated in suspension for 2 hours (time periods 0-28 hours tested) after which the aggregates were transferred to a glass centrifuge tube with the 80 ml brought to the final medium volume. Alternatively, the cell suspension can be directly placed in a centrifuge tube stirred at 55 revolutions per minute or an Erlenmeyer flask stirred at 40 revolutions per minute, and the clusters formed in the stirred suspension (Figure 3c) are brought to a final medium volume of 80 ml.

Using this sequential passaging method, cells were passaged 20 times, with an approximate multiplication rate of 1: 3 seeding in each passage. Pluripotency was measured at each passaging using flow cytometry, and karyotyping was performed using in-situ fluorescent hybridization analysis (FISH) for chromosomes 12 and 17; two chromosomes are identified as potentially unstable in hES cells. Flow cytometry results for CD9, SSEA4, TRA-1-60, TRA-1-81 and CXCR4 expression are shown in graph form and show a high expression level of pluripotency markers and low or absent differentiation marker expression (CXCR4), while FISH analyzes for chromosomes 12 and 17 showed a normal number of copies. These data show that H1 hES cells can be stored in suspension culture after standard serial passaging using Accutase ^® , applying the enzymatic method of cell dissociation using enzymes not originating from mammals, and will maintain pluripotency and stable karyotype in a dynamic suspension culture system, when generating 1 × 10 ⁹  cells for each source cell for 20 passages. EDTA can also be used for this series of suspensions in 6 passages.

Table 3
The results of flow cytometry on cell pluripotency markers, shown as a function of time based on results for markers CD9, SSEA4, TRA-1-60, TRA-1-81 and CD184 (CXCR4) Passage
(culture day) CD9 SSEA4 TRA-1-60 TRA-1-81 CD184 13) 92.0% 100.0% 57.4% 58.6% 0.2% 2 (4) 73.3% 99.9% 63.5% 54.3% 0.1% 3 (3) 87.5% 99.7% 65.8% 63.6% 0.1% 4 (4) 86.7% 99.8% 60.9% 68.2% 0.1% 5 (3) 79.3% 99.7% 67.6% 69.9% 0.3% 6 (3) 79.3% 99.7% 67.6% 69.9% 0.3% 7 (3) 93.7% 100.0% 60.1% 58.8% 0.2% 8 (3) 83.0% 99.0% 73.0% 68.0% 0.5% 9 (4) 94.6% 100.0% 65.5% 64.2% 0.1% 10 (4) 96.3% 100.0% 77.3% 75.0% 0.2% 11 (4) 97.3% 100.0% 69.1% 61.3% 0.2% 12 (4) 91.6% 100.0% 56.9% 62.7% 0.6% 13 (4) 97.3% 99.9% 62.9% 63.2% 0.2% 14 (4) 97.1% 100.0% 71.1% 82.4% 1.0% 15 (4) 96.1% 99.6% * 79.0% 74.2% 0.2% 16 (4) 87.7% 99.9% 77.1% 72.5% 0.3% 17 (4) 98.6% 99.7% 69.9% 57.7% 0.3% 18 (4) 97.7% 100.0% 68.6% 56.6% 0.2% 19 (4) 97.1% 100.0% 79.4% 70.4% 0.1% 20 (4) 96.9% 100.0% 57.4% 55.7% 0.4%

EXAMPLE 4

DIRECTED DIFFERENTIATION OF SUSPENSION CULTURE OF HUMAN EMBRYONIC STEM CELLS OF H1 CELL CELL LINE

Cell line of human embryonic stem cells H1 (WA01 cells, WiCell, Madison, Wisconsin, USA) at 40 passaging were removed from culture planar attached using Accutase ^® and transferred to suspension culture format. The cells were maintained in a dynamic suspension culture system for 30 passages using the method described in example 3.

Pluripotency was confirmed during the first 20 passages, as shown in Table 3, with consistently high levels of pluripotency markers that were maintained throughout the culture, as determined by flow cytometry. To confirm pluripotency and demonstrate the ability of a cell source to treat diabetes, cells were differentiated into pancreatic progenitor cells in a dynamic suspension culture system using a stepwise progression method in various media containing morphogens or growth factors intended for normal pancreatic development. This process leads to the emergence of a population of pancreatic progenitor cells, which are characterized by high co-expression of Pdx1 and Nkx6.1. When these cells were transplanted, they further matured into tissues capable of glucose-stimulated insulin secretion, capable of secreting insulin in response to glucose and maintaining normal blood glucose levels in a streptozotocin-induced diabetes model. See FIG. 4c and table 4c.

To generate these progenitor cells of the pancreatic cells, human H1 embryonic stem cells, which were grown and maintained in a dynamic suspension culture system for 16 passages, were differentiated according to the method described in example 3. Thus, the cells were grown for 30 Passages tested for pluripotency during the first 20 passages; cells differentiated at passage 16. Pluripotent cells in cluster format were transferred from mTeSR®1 medium to FBC solution (Table 4a) for 3 hours at 4 ° C. The cell clusters were then transferred to a suspension in a 3-liter glass bioreactor, controlled by a Sartorius Stedim BioStat B-DCU control unit (Goettingen, Germany) and suspended in a differentiation medium in the amount of 0.55 cells / ml in accordance with Table 4b The cells were maintained for 14 days in a closed sterile suspension in a bioreactor with adjustable temperature indicators, pH and dissolved oxygen (DO) (FermProbe ^® pH electrode is 225 mm, the model № F-635, a sensor of dissolved oxygen OxyProbe ^® 12 mm, model number D-145 from Broadley-James Corporation , Irvine, California, USA).

During the run, the bicarbonate levels of the medium were maintained at 3.64 g / l, the pH was maintained at a pH of 7.4 by regulating the flow of CO2 in a total volume of medium ≤1.6 l The bioreactor void was continuously treated with CO2, air and O2 under the control of a Sartorious control unit with a set dissolved oxygen level of 20% at stage 1 and a set dissolved oxygen level of 30% at stage 2 followed by a constant gas flow of 150cc / min. The flow of oxygen is regulated relative to the content of dissolved oxygen and the flow of CO2 is regulated relative to the pH level. The temperature was maintained at 37 ° C during the entire run of an electric heated casing. At the beginning of the run and for each medium change (93% of the medium was removed by replacing), the blades (3 ”, threaded paddle wheels made of stainless steel operating at 70 revolutions per minute) were stopped and the medium was removed or added using peristaltic pump through a submersible tube in a bioreactor connected to a C-Flex® tube using a Terumo ™ pipe welding mill to maintain a closed system. Images of cells / clusters were obtained at the end of each stage of differentiation, and the samples subjected to flow cytometry were collected and analyzed for the expression of CXCR4 at stage 1 for 3 and 3 days at the end of stage 2 (Figure 4a). There was an almost complete transition of the population from the CXCR4-negative pluripotent cell population at the beginning of differentiation (table 3, channel 16) to the population of CXCR4-positive (98.5% of CXCR4-positive cells, Figure 4B) cells. These cells then moved to a near-CXCR4-negative state after 3 days at the end of stage 2 (7.0% of cells were CXCR4-positive), and by the end of stage 3, the cells almost completely went into a CD56-positive state. At the end of the process of differentiation on the 4th day of the 4th stage, the cells were 88.5% Pdx1-positive (4b) and showed expression patterns corresponding to a combination of pancreatic endocrine cells (33.5% chromogranin-positive) and pancreatic cells- predecessors (65.7% Nkx6.1-positive). Patterns of marker expression characteristic for this stage indicate an effective stepwise differentiation from the population of pluripotent cells into pancreatic cells. At the end of the differentiation process, 2.77 million cells / ml (4.1 billion cells in 1.5 l) was obtained, indicating a full growth of 5 differentiated cells per each HES cell line injected.

In the end, 500 ml were removed by centrifugation and washing and were tested on animal models of engraftment, maturation and functioning. The remaining 1000 ml of the cell suspension was treated with the kSep ^® 400 system (KBI Biosystems, Durham, North Carolina, USA) for washing, filtering and concentrating the cell product in a fully closed system. The cell product is concentrated from an initial volume of 1 l in 50 ml of concentrated cells to a final concentration of 41 million cells / ml. These concentrated cells are then poured into 24 vials, 1.2 ml each, using an automated vial filling machine (Fill-It, TAP, Hertfordshire UK) and frozen by placing in a refrigerator with liquid nitrogen.

500 ml of differentiated cells, which were washed and concentrated by standard centrifugation, were transplanted at a dose of 5 million cells per mouse SCID-Bg line directly below the renal capsule, or placed inside immuno-protective macro-encapsulation devices (TheraCyte ™, Irwin, California, USA), which were implanted subcutaneously (6 animals for each condition). 12 weeks after implantation, the implanted cells expressed significant levels of circulating human C-peptide (> 0.1 ng / ml), which was determined by ELISA (ELISA of the human C-peptide, Mercodia catalog number 10-1141-01) in response on starvation and subsequent feeding and after 16–20 weeks, animals showed more than 1 ng / ml of circulating C-peptide (Table 4c).

After 27 weeks (190 days) after implantation, two animals from the device with encapsulated immune-protective grafts received one large dose of high streptozoticin (STZ) to selectively destroy all endogenous mouse islet β-cells and induce diabetes (250 mg / kg). After the next two weeks after STZ treatment, sufficient to induce overt diabetes in control animals, blood glucose levels in animals with tangled grafts remained within the normal range (<150 mg / dL). At 29 weeks after implantation and two weeks after the introduction of STZ, the two animals were then tested for glucose-stimulated insulin secretion (GSIS) and showed a marked increase in the circulation of the human c-peptide in response to the introduction of glucose. In addition, when each of the grafts were removed at 209 days (29.5 weeks) after implantation, the blood glucose level of the animals significantly increased to> 500 mg / dl.

These results indicate that a product isolated from human embryonic stem cells and intended to treat diabetes can be obtained from a suspension of grown and differentiated stem cells. The product can be generated in a scalable, closed-loop bioreactor mixing system and the cell product can be processed by washing in a closed system and concentrated, as required for commercial production of cGMP. The product, isolated from human embryonic stem cells, is suitable for treating diabetes in a widely used animal model of diabetes, which is confirmed by GSIS competencies, has the ability to regulate blood glucose levels, and shows the return of the diabetic state after removal of cell therapy.

Table 4a
The composition of the FBC solution Component Amount (mg / l) Function Level Anhydrous Dextrose 901 Sugar United States of America Potassium chloride 559 salt United States of America Bicarbonate of soda 2000 Buffer solution United States of America Sucrose 6846 Sugar United States of America Mannitol 3644 Sugar alcohol United States of America Calcium chloride dihydrate (CaCl ₂ .2H ₂ O) 70 salt United States of America Magnesium Chloride (MgCl ₂ .6H ₂ O) 1017 salt United States of America Potassium bicarbonate (KHCO ₃ ) 500 Buffer solution United States of America Potassium monosulfate (KH ₂ PO ₄ ) 1361 Buffer solution NF ^b / FCC ^c Lactobionic acid 35830 Cell stabilizer N / D ^d L-glutathione 922 Antioxidant N / A HCl For pH adjustment Acid ACS ^e Sodium hydroxide For pH adjustment The foundation NF / FCC Water for injections To prepare the solution To prepare the solution United States of America ^a USP = United States Pharmacopoeia
^b NF = National Formulary
^c FCC = Food Chemical Codex
^d N / A = No data
^e ACS = United States Chemical Society

Table 4b
Environment components and differentiation protocol Stage 1 Stage 2 Stage 3 Stage 4 Base environment
(final glucose concentration) MCDB131
3.64 g / l NaCO ₃
(8 mM glucose)
2% fatty acid free bovine serum albumin (FAF-BSA)
and 2mM L-glutamine Protein / amino acid supplements Growth factors
AND / OR
Small molecules MCX (3 μM)
0-24 hours
GDF8 (100 ng / ml) for 24-72 hours
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1: 200)
RA (2µmol)
SANT (0.25 μM)
AA (5 ng / ml)
TppB (200 nM)
LDN (100 nmol) on
0-24 hours stage 3 ITS-X (1: 200)
SANT (0.25 μM)
Cypi (100 nM)
SCIO (2μM)
TppB (100 nM) Total day 3 3 3 five Medium change Time 0 and 24 hours Time 0 and 48 hours Time 0 and 24 hours Time 0, 48 and 96 hours

(Nomenclature Time 0 = first seeding in a new stage; time 24, 48 or 96 hours = time after changing the medium of the stage)

Table 4c
 Expression of C-peptide (ng / ml) C-peptide (ng / ml) 4wk 8wk 12wk 16wk 20wk 24wk 29wk Implant in the renal capsule (N = 6) 0.00 0.03 0.19 0.95 2.56 STDEV 0.00 0.03 0.17 0.71 1.33 Implanted Theracyte Device (N = 6) 0.00 0.02 0.35 0.58 1.45 2.49 2.85 ST. OFF 0.01 0.01 0.54 0.51 1.02 0.75 0.21

EXAMPLE 5

DIRECTED DIFFERENTIATION IN THE FORMAT OF SUSPENSION CULTURE OF ATTACHED HUMAN EMBRYONIC STEM CELLS OF H1 CELL CELL LINE

Human H1 embryonic stem cell lines (WA01, WiCell, Madison Wisconsin cells), with passaging 41, were removed from a flat attached culture using EDTA and transferred to the suspension culture format according to the method described in Example 2.

Cell aggregates pluripotency was measured using flow cytometry, as shown in Fig. 5a, with high expression of the pluripotency markers CD9, SSEA4, TRA-1-61, TRA-and 1-80, indicating a high level of cell pluripotency. These pluripotent cells are then differentiated into pancreatic progenitor cells in a dynamically mixed suspension culture system using a stepwise progression method in various media containing small molecules and growth factors designed for normal pancreatic development. This process produces pancreatic progenitor cell populations characterized by the coexpression of pancreatic transcription factors Pdx1 and Nkx6.1. When these cells were transplanted, they further matured into tissues capable of glucose-stimulated insulin secretion, which can correct the high level of blood glucose in the streptozotocin-induced diabetes model.

To generate a population of pancreatic progenitor cells, pluripotent cells in cluster format, supported in the mTeSR®1 medium, were transferred to a 0.2-liter suspension stirred glass bioreactor (Dasgip, DS0200 TBSC, Shrewsbury, Massachusetts, USA a) with adjustable controller temperature, pH and dissolved oxygen level. Pluripotent cell clusters were cultured in the bioreactor for two days. At this time (stage 1, day 0) the medium was replaced and differentiation was initiated, the cell aggregates were suspended in an amount of about 0.7 million cells / ml in the medium for differentiation in accordance with Table 5a. The cells were then kept in this closed sterile suspension in the bioreactor for 14 days. During differentiation, the levels of bicarbonate medium were maintained at 3.64 g / l, the pH was maintained at 7.4 by regulating the flow of CO2 in the total volume of the medium 0.3 l. The free space of the bioreactor was continuously treated with CO2 and air under the control of the Dasgip control system with a fixed level of dissolved oxygen of 30% at a constant gas flow of 5 l / h. The air flow is regulated relative to the dissolved oxygen content and the flow of CO2 is regulated relative to the pH level.

Table 5a
Environment components and differentiation protocol Stage 1 Stage 2 Stage 3 Stage 4 Base environment
(final glucose concentration) MCDB131
3.64 g / l NaCO ₃
(8 mM glucose) MCDB131
3.64 g / l NaCO ₃
(8 mM glucose) MCDB131
3.64 g / l NaCO ₃
(8 mM glucose) MCDB131
3.64 g / l NaCO ₃
(8 mM glucose) Protein supplements 2% fatty acid free bovine serum albumin (FAF-BSA)
and 2mM L-glutamine Growth factors
AND / OR
Small molecules MCX (3 μM)
As noted
GDF8 (100 ng / ml)
As noted
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1:50 000) FGF7 (50 ng / ml)
ITS-X (1: 200)
RA (2 μM)
SANT (0.25 μM)
AA (5 ng / ml)
TppB (200 nM)
LDN (100 nmol) for 0-24 hours, stage 3 ITS-X (1: 200)
SANT (0.25 μM)
Cypi (100 nM)
SCIO (2µM)
TppB (100 nM) Total day 3 3 3 five Medium change As noted Time 0 and 48 hours Time 0 and 24 hours Time 0, 48 and 96 hours

In this example and in the description, SCIO is an Alk5 inhibitor with the chemical name 4 - {[2- (5-chloro-2-fluorophenyl) -5- (1-methylethyl) pyrimidin-4-yl] amino} -N- (2 -hydroxypropyl) pyridine-3-carboxamide and CAS No. 674794-97-9. The chemical structure of SCIO is presented below:

At this stage, the temperature was maintained at 37 ° C. At the beginning of the run and for each medium change (95% of the medium was removed by replacing), the blade was stopped and the medium was removed, added using a peristaltic pump through a dip tube in a bioreactor connected to a C-Flex® tube using a Terumo ™ tube welding mill to maintain a closed system.

Several different feed settings were tested during stage 1: (a) medium change 24 hours after differentiation began, without medium change after 48 hours; (b) replacing the medium 24 hours after the start of differentiation and adding a bolus of glucose after 48 hours; and (c) without changing the medium during stage 1 with the addition of glucose bolus and GDF8 24 hours after the start of differentiation and the subsequent addition of glucose bolus 48 hours after the start.

The number of cells at the start, middle and end of the process, taken from each reactor, is shown in Table 5b. At the end of stage 1, cells were selected to determine protein expression patterns using flow cytometry. Cells differentiated by Condition A — replacement of the medium 24 hours after the start of differentiation to the definitive endoderm, then without changing the medium for the next 48 hours — showed better results in measuring the induction of differentiation markers (CD99 and CXCR4) and reducing the expression of the pluripotency marker (CD9 ) (5 B). Higher expression of CXCR4 and CD99 in combination with low CD9 expression at the end of the final endoderm formation correlates with higher expression of pancreatic genes and low expression of genes showing the development of alternative organs in the late stages of differentiation (Fig. 5d and 5e). In particular, the absence of medium substitution during the first stage of differentiation or the addition of glucose to the medium in stage 1 in the volume feed format resulted in a decrease in CXCR4 levels at the end of stage 1, which correlated with a different aggregate morphology at the end of the fourth stage of differentiation (Fig. 5c). In particular, conditions B and C had lower pancreatic gene expression (Nkx6.1 and CHGA) and higher expression of non-pancreatic genes (CDX2 and Sox2) at the end of stage 4, as measured by flow cytometry (Fig. 5d and Table 5b). These findings were confirmed by qRT-PCR (Fig. 5e), since condition A showed significantly higher expression of pancreatic genes than condition C, condition B gave results averaging between A and C. In addition, condition C showed significantly higher levels of indicative genes of alternative non-pancreatic development, for example, CDX2, AFP and albumin (Fig. 5e). These data show that the homogeneous definitive endoderm (DE) with a high level of CXCR4 expression, which is generated without changing the medium in the last 48 hours of DE formation, can later turn into a pure population of pancreatic endoderm.

At the end of stage 4 differentiation, cells differentiated according to Condition A were removed from the bioreactor, washed in MCDB131 medium containing 0.1% FAF-BSA, and implanted into SCID-BG mice. Each mouse was transplanted 5 million cells directly under the renal capsule. Every 4 weeks after implantation, blood samples were taken and glucose and C-peptide levels were measured in the blood. 12 weeks after implantation, the human C-peptide was detected at levels above 1 ng / ml, and after 16 weeks the levels of C-peptide averaged 2.5 ng / ml. (fig.5f). At week 20 after implantation, the level of C-peptide was measured in animals in a hungry and then fed state. Glucose treatment caused a significant increase in circulating human C-peptide from 0.93 ng / ml in a hungry state to 2.39 ng / ml in a full-bodied state (figure 5 g), indicating that the transplanted cells had matured into a functional GSIS -competent fabric. In addition, when animals were injected with streptozotocin (STZ) (mouse β-cells are more susceptible to STZ and more destroyed than human β-cells) to trigger a diabetic condition, animals with a graft of functional GSIS-competent tissue maintained normal blood glucose levels in contrast to untreated control animals that developed overt diabetes (Fig. 5h). These results indicate that animals with a HES-differentiated cell transplant were protected from STZ-induced diabetes with the help of a functional graft of pancreatic tissue.

Table 5b
Cell count and flow cytometry data Pluripotency
(Condition) The density of viable cells (million cells / ml) CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 (a) 0.723 93,8 0.2 100 74.3 67.3 (B) 0.677 92.3 0.2 100 71.7 71 (C) 0.738 89.9 0.1 100 75.3 72.1 DK
(Condition) The density of viable cells (million cells / ml) CD9 CD184 CD99 (a) 0.965 1.7 99.6 84.3 (B) 1.22 4.8 93.1 81.2 (C) 1.2 8.3 68 34.1 PC
(Condition) The density of viable cells (million cells / ml) NKX6.1 Synaptophysin CDX2 SOX2 NKX2.2 CHGA (a) 0.795 47.5 48.4 2.9 23.8 61.7 55.7 (B) 0.98 44.4 38.5 10.3 21.4 45.4 41.5 (C) 1.33 15.4 5.8 37 18.4 9.6 6.7

EXAMPLE 6

DIRECTED DIFFERENTIATION IN THE FORMAT OF SUSPENSION CULTURE OF MICRONS CARRIERS WITH ATTACHED HUMAN EMBRYONIC STEM CELLS OF H1 CELL LINE

Cytodex ^® 3 (C3) microcarrier spheres (Sigma-Aldrich Co LLC, St. Louis, Missouri, USA, catalog number C3275) were prepared for culture by soaking 400 mg of spheres in 20-ml silicon-coated glass scintillation vials containing 15 ml of Dulbecco's PBS (DPBS), for 4-24 hours. Cytodex ^® 3 consists of a thin layer of denatured collagen, chemically bound to a matrix of cross-linked dextran. The layer of denatured collagen on Cytodex ^® 3 carriers can be decomposed by various proteases, including trypsin and collagenase, which makes it possible to extract cells from the microcarrier, while maintaining maximum viability, functionality and integrity of the cells.

After soaking, the spheres were autoclaved, washed with sterile DPBS, and resuspended in the conditioned medium of mouse embryonic fibroblasts (MEF-CM) with the addition of 10 μM Y-27632. The spheres are then transferred to 125 ml glass centrifuge tubes Corning ^® (Corning Incorporated, Corning, NY, USA) at a density of 100 mg of spheres / flask. Centrifuge tubes containing spheres and MEF-CM with Y-27632 were equilibrated in a humidified 5% CO2 incubator at 37 ° C for at least 60 minutes.

Human H1 embryonic stem cell lines (WA01 cells, WiCell, Madison, WI, USA), when passaged 44 were removed from a flat attached culture using TrypLE ™ (Life Technologies Corporation, Grand Island, NY, USA) (8-minute incubation at 37 ° C with the formation of a suspension of individual cells). The cells were then washed and suspended in MEF-CM with Y-27632 and 11 million hES cells attached to spheres for 6 hours during the static incubation period. Then MEF-CM with Y-27632 was added to the centrifuge tube, bringing the final volume of the carrier to 75 ml, and the cells and spheres were mixed in a glass centrifuge tube at a blade speed of 50 revolutions per minute. Cells were grown in this way for 5 days with daily replacement of 50 ml of MEF-CM medium. After 5 days in culture, the flasks contained 53 × 10 ⁶ cells (± 12 × 10 ⁶ WITH). As a control, one million H1 HES cells were also seeded on the bottom of a 6-well polystyrene Petri dish, coated with Matrigel ™ diluted 1:30, and maintained with a daily MEF-CM medium change.

After 5 days in a pluripotent culture, these cells were differentiated into pancreatic progenitor cells in a dynamic suspension culture system by stepwise progression in various media containing one or both of small molecules and growth factors intended for the recapitulation of normal pancreatic morphogenes. Two medium formulations were tested as a way to maintain recapitulation of the normal development of the pancreas; one using activin A and Wnt3a to form DE, and the other using MCX compound with GDF8 to form DE (tables 6a and 6b, respectively). The medium was changed daily and the samples were characterized by RT-PCR and flow cytometry to determine cell properties. Phase-contrast images of cells on microcarriers were obtained, and a graph of cell morphology in pluripotent cultures over time until the initiation of cell differentiation is shown in FIG. 6a. A time chart showing the differentiation of cultures is shown in FIG. 6b. It was also determined the number of cells taken at different points in time during the experiment, and the results are presented depending on the surface area (cells / cm ² in Fig. 6C) or the volume of the carrier (cells / ml in Fig. 6d) for medium preparations on a flat culture or suspended culture on microcarriers.

Cells are characterized at various points throughout the process by flow cytometry and RT-PCR. The results of flow cytometry of the first stage of differentiation and the formation of the definitive endoderm are shown in the form of a dotted diagram of cell expression of CXCR4 (Y axis) and CD9 (X axis) in FIG. 6e, also shows the total expression of each marker in FIG. 6f. The results show that under any conditions, the vast majority of cells form the definitive endoderm, which is determined by the gain of CXCR4 expression and the loss of expression of the surface marker of pluripotency CD9. In addition, the most efficient formation of definitive endoderm occurs in descending order when processed as follows: microcarriers MCX / GDF8> flat culture MCX / GDF8> microcarriers WNT3A / AA> planar culture WNT3A / A.A. This is how the specific effect of the medium on the cells manifests itself, since the cells treated with MCX / GDF8 show low expression of CERBERUS (Cer 1), GOOSECOID and FGF17 (Fig. 6g). However, all treatment conditions show similar levels of gene expression of definitive endoderm; CD99, CXCR4, FOXA2, KIT and SOX17 (Fig. 6g and Table 6c). These processes generate populations of pancreatic progenitor cells characterized by the co-expression of pancreatic cell transcription factors, Pdx1 and Nkx6.1. When these cells were transplanted, they further matured into tissues capable of glucose-stimulated insulin secretion, which can correct the high level of blood glucose in the streptozotocin-induced diabetes model.

Below, in Table 6a, B27 is an additive of Gibco® B-27® (Life Technologies Corporation, Carlsbad, California, USA).

As used in this example, the MCX compound is 14-prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ 0.1 ~ 8.12. ~] heptacosis-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-non-an-16-one, which has the following formula (formula 1):

Other cyclic aniline-pyridinotriazines can also be used instead of the MCX compound described above. Such compounds include, but are not limited to, 14-methyl-3,5,7,14,18,24,28-heptaazatetracyclo [20.3.1.1 ~ 2.6 ~ .- 1 ~ 8.12 ~] octacose-1 (26) , 2 (28), 3,5,8 (27), 9,11,22,24-nona-17-one-e and 5-chloro-1,8,10,12,16,22,26, 32 -octaazapentacyclo [24.2.2.1 ~ 3.7 ~ -1 ~ 9.13 ~ 0.1 ~ 14.18 ~] tritriacont-3 (33), 4.6.9 (32), 10, 12.14 (31 ), 15,17-nonaen-23-he. These compounds are listed below (formula 2 and formula 3):

Examples of suitable compounds are described in US patent application US No. 2010/0015711, the disclosure of which is included in its entirety as it relates to MCX compounds associated with cyclic aniline-priridinotriazinah and their synthesis.

The Cyp26 inhibitor used in this example in step 4 was N- {4- [2-ethyl-1- (1H-1,2,4-triazol-1-yl) butyl] phenyl} -1,3-benzothiazole- 2-amine having CAS No. 201410-53-9 and the following structure.

This Cyp26 inhibitor is also known as Cypi. The structure and synthesis of this Cyp26 inhibitor is described in US Pat. No. 7,378,433, the disclosure of which is incorporated by reference in its entirety, as it relates to Cyp26 inhibitors and their synthesis.

Table 6a
Medications and differentiation protocol Stage 1 Stage 2 Stage 3 Stage 4 Base environment RPMI
11 mM glucose DMEM / F12
17.5 mM glucose DMEM
25 mM glucose Supplements + 0.2% FBS + 0.5% FBS + 2% FBS + 1% B27 Growth
Indicator
And / or
Small
molecules AA
(100 ng / ml)
Wnt3a
(20 ng / ml) AA
(100 ng / ml) FGF7
(50 ng / ml) Noggin
(100 ng / ml)
PP
(2µM)
SANT1
(250nM) Noggin
(100 ng / ml)
ALK5i
(1 µmol)
TPB (50 nM) Noggin
(100 ng / ml)
ALK5i
(1 µmol) Day 1d 2d 3d 4d 4d 2d

Table 6b
Medications and differentiation protocol Stage 1 Stage 2 Stage 3 Stage 4 Base environment MCDB131
8 mM glucose MCDB131
10.5 mM glucose MCDB131
25 mM glucose MCDB131
25 mM glucose Supplements 2% FAF-BSA 2% FAF-BSA 0.1% AlbuMAX® 0.1% AlbuMAX® Growth
growth
Small molecule agonist /
antagonist GDF8
100 ng / ml
MCX (day 1 only)
3 µmol
1: 50,000 ITS-X FGF7
50 ng / ml
1: 50,000 ITS-X FGF7 (50 ng / ml)
AA (5 ng / ml)
RA (2 μM)
SANT (250 μM)
LDN 193189
1: 200 ITS: X PKC activator (500 nM)
SANT (250 nM)
LDN 193189 (200 nM)
Cyp26 inhibitor (100 nM)
1: 200 ITS: X Day four 3 four 6

Table 6c Description Reference H1 hES WNT3A / AA FLAT WNT3A / AA microcarrier MCX / GDF8 FLAT MCX / GDF8 microcarrier GAPDH control one one one one one AFP one 0.6 0.0 4.7 0.0 CD9 one 1.0 0.9 0.3 0.5 CD99 one 10.5 10.9 18.5 7.1 CDH1 one 1.2 0.6 0.5 0.6 CDH2 one 24.8 28.4 47.8 27.8 CDX2 one 23.2 0.0 74.9 27.8 CER1 one 346.2 649.7 8.1 5.6 CXCR4 one 280.3 190.1 153.9 154.7 FGF17 one 1406.4 3174.5 92.0 112.9 FGF4 one 0.8 0.5 0.0 1.1 FOXA2 one 432.5 424.3 588.5 321.2 Gata4 one 252.4 165.3 1100.1 444.9 GATA6 one 607.1 939.9 709.4 312.0 GSC one 49.0 81.6 0.3 0.6 KIT one 16.3 17.9 12.3 8.0 MIXL1 one 33.2 95.6 16,0 19.1 MNX1 one 146.3 111.4 595.8 392.6 Nanog one 0.4 0.5 0.0 0.2 OTX2 one 22.9 26.4 9.1 8.3 OCT4 one 1.5 1.1 0.0 0.5 SOX17 one 751.1 1198.2 1235.0 796.3 SOX7 one 0.6 1.7 5.5 0.7 T one 64.1 7.1 22.3 212.9

Example 7

A sub-clone of the cell line H1 (WA01) hES - WB0106 is used for this example. Sub-clone WB0106 was obtained at the WiCell Research Institute (Madison, WI) from a seed from the H1 line, called DDL-13. A sub-clone of the WB0106 H1 line was obtained from a vial of DDL-13 thawed by passaging 23 in mTeSR ^® 1 on Matrigel ™ substrate, and then passaged with EDTA. WB0106 was frozen by passaging 28 and was selected for these studies based on the normal karyotype (FISH and G-bands), the ability to differentiate into pancreatic progenitor cells and the competence to form clusters and grow in suspension culture.

The WB0106 WCB bottle was then thawed in medium on Matrigel ™ substrate in a T225 flask (Corning Incorporated, Corning, New York, USA) and, when first passaged, the cells were divided into several T225 flasks. On the second passaging, cells from several T225 flasks were combined and used to fill one Cell Stack ™ single bilayer cell (CS2). After forming a confluent layer in CS2 by 70%, a system of tubes with C-Flex® caps was attached to the ports of the medium, to which tubes leading to the pump were connected to close the system. After the system was closed using C-Flex® tubes, bags or bottles were welded to it using the Terumo machine, where fluid volumes (medium, PBS ^{- / -} , Accutase ^®, or suspended cells) were transferred with a peristaltic pump.

For separation of CS2 cells, the cells were washed once with PBS ^{- / -,} then treated with a solution of half Accutase ^® efficiency diluted PBS ^{- / -} and incubated for 4-5 minutes. Then Accutase ^® was removed and after 3 minutes after application of the enzyme solution, CS2 was used to stimulate cellular compartment. The vial with the medium supplemented with 0,5% BSA, containing 10 mmol / l of Rho-kinase inhibitor, Y-27632, was pumped into the washing and CS2 to inactivate residual Accutase ^®, whereupon wash was collected. A second wash volume was added, the wash was collected and combined with the first. Then, 2.0-2.5 × 10 ⁸ cells in 200 ml were transferred to a CellSTACK® single-layer cell container and incubated at 37 ° C for 2 hours in a humidified incubator with 5% CO ₂ . Using a closed loop C-Flex® tube with a pump tube attached between the two ports of the CellSTACK® medium, the cell suspension was ground for 5 minutes at 75 revolutions per minute using a peristaltic pump to homogenize the aggregates. The closed loop of the tubes was replaced with 0.2 micron sterile filters for gas exchange and the CellSTACK® was incubated overnight at 37 ° C in a humidified 5% CO ₂ incubator. After incubation overnight (12–22 hours, 18 hours optimally), the cells in CellSTACK® form rounded spherical aggregates (clusters) of iPS cells.

Medium supplemented with 0.5% BSA containing suspended cell clusters was transferred from CellSTACK® to a disposable 1-liter centrifuge tube (Corning; Corning, New York, USA) along with 0.4 l of fresh medium, supplemented with 0 , 5% BSA and kept 55-65 revolutions per minute. Twenty-four hours after transfer, a disposable 1-liter centrifuge tube was removed from the humidified 5% CO2 incubator and the clusters were allowed to settle for 5-10 minutes. Then the medium was aspirated until 200 ml remained and 400 ml of additional fresh culture medium was added to the centrifuge tube. This process was repeated at the end of 2 days (48 hours after transfer).

At the end of 3 days (72 hours after transfer to the centrifuge tube with CS2), cell clusters were treated with Accutase ^® for passaging and further growth. The passaging process was initiated by removing 1 liter of a disposable centrifuge tube from a humidified 5% CO ₂ incubator. The tube was placed in a biologically safe centrifuge box to maintain a homogeneous suspension of cells. After that, the cell suspension was removed from the centrifuge tube with a 100 ml pipette, evenly distributed between four 175 ml conical polycarbonate tubes (ThermoFisher-Nalgene; Buffalo, New York, USA) and centrifuged for 5 minutes at 80-200 OCS . The spent medium was aspirated without disturbing the cell pellet. Then to each tube was added 25 ml of DPBS without calcium or magnesium (DPBS ^{- /} ), and the cells were combined into one conical tube and centrifuged for 5 minutes at 80-200 OCS. DPBS ^{- / was} aspirated from a conical tube and 30 ml of 50% Accutase ^® / 50% DPBS ^{- /} solution was added to the tube. Cell clusters were pipetted 1-3 times, and then periodically mixed for 4 minutes, then centrifuged for 5 minutes at 80-200 OCS. Accutase ^{® was} then aspirated as completely as possible without disturbing the cell pellet, and the conical tube was tapped continuously and gently for 3-5 minutes until the cell suspension became uniform milky white. 10 ml of medium supplemented with 0,5% BSA, containing 10 mmol / l of Rho-kinase inhibitor, Y-27632 was added to the cell suspension and milled 2-4 times to inactivate residual Accutase ^®. 90 ml of medium supplemented with 0.5% BSA containing 10 μmol / l Rho kinase inhibitor, Y-27632, was added to the cells and the suspension was passed through a 40 μM cell sieve (BD Falcon; Franklin Lakes, NJ , USA).

The cell density in a volume of 100 mL of the filtered cell suspension were determined using the apparatus NucleoCounter ^® NC-100 (ChemoMetec A / S, of Allerod, Denmark) was added and an additional medium to a final cell concentration of 1 × 10 ⁶ cells / ml in medium supplemented with 0 , 5% BSA containing 10 µmol / l Rho kinase inhibitor, Y-27632. Then, 225 ml (225 million cells) of the cell suspension was transferred to a 1-liter rotating disposable vessel and incubated for 1 hour without stirring in a humidified 5% CO2 incubator. Then the flask was removed from the incubator and stirred at 100 revolutions per minute on a rotating plate in a biologically safe box for 1-3 minutes. While the cell suspension was mixed, in addition to the cell suspension, 225 ml of medium was added, supplemented with 0.5% BSA containing 10 μmol / L of Rho-kinase inhibitor, Y-27632. The centrifuge tube was then returned to a humidified 5% CO2 incubator for 30 minutes. Then the flask was removed from the incubator and stirred at 100 revolutions per minute on a rotating plate in a biologically safe box for 1-3 minutes. While the cell suspension was mixed, in addition to the cell suspension, 150 ml of medium was added, supplemented with 0.5% BSA containing 10 μmol / L in Rho-kinase, Y-27632, to bring the final volume to 600 ml, and the flask was returned to stirred suspension in an incubator. After 24 and 48 hours after the dissociation of Accutase ^® , the cell clusters were allowed to settle to the bottom of the flask for 5-10 minutes. To ensure that the loss of clusters is minimized, 400 ml of the spent medium was removed by aspiration from the flask and replaced with fresh medium. Using this process, H1 cells were transformed from an attached culture onto clusters of cells in suspension culture.

72 hours after the initial Accutase ^® treatment, the process of cell dissociation of clusters and seeding (passaging) in a centrifuge tube was repeated to keep the cells in suspension for repeated passaging (test range: 1-10 passages). The process described above was repeated, with the difference that after the first 24 hours the medium was not removed, but 200 ml of fresh medium was added. 48 hours after the dissociation of Accutase ^® , the cell clusters were allowed to settle to the bottom of the flask for 5-10 minutes, 600 ml of medium were aspirated and 400 ml of fresh medium was added to the flask.

These cells, which have undergone suspension and culture, can then be cryopreserved and stored for future use. In order to prepare a suspension of overgrown cells for cryopreservation, cell clusters were dissociated with Accutase ^® , as described above for passaging the suspension, except that the cells did not pass 40 microns through the cell filter. It was determined the number of cells in 100 ml of cell suspension, created from a solution in each disposable 1-liter flask. The cell suspensions were then combined and centrifuged for 5 minutes at 80-200 OCS. The medium from the centrifuge tube was then removed as completely as possible, without disturbing the cell pellet. Cold (<4 ° C) CryoStor®10 (Stem Cell Technologies, Inc., Vancouver, British Columbia, Canada) was then added dropwise so as to reach a final concentration of 150 million cells per ml, and the cell solution was kept on ice a bath during transfer to a 1.8 ml Corning® cryopreservation bottle (Corning Incorporated, Corning, New York, USA) or a 15 ml Miltenyi cryoconservation bag (Miltenyi Biotec Inc. Auburn, California, USA) .

The suspension of overgrown cells was then frozen in a vial at high density in a freezer at a controlled rate as follows. The chamber was pre-cooled to 4 ° C and the temperature was maintained until the test tube with the sample reached 6 ° C. Then the temperature in the chamber was lowered at a rate of 2 ° C / min until the test tube with the sample reached -7 ° C. After the sample tube reached -7 ° C, the chamber was cooled at 20 ° C / min to -45 ° C. After that, the temperature in the chamber was raised by 10 ° C / min until the temperature in the chamber reached -25 ° C, and then the chamber was further cooled at 0.8 ° C / min until the test tube with the sample reached temperatures of -45 ° C. The temperature in the chamber was then reduced by 35 ° C / min until the temperature in the chamber reached -160 ° C. The temperature in the chamber was then kept at -160 ° C for at least 10 minutes, after which the tubes were transferred to a storage facility with liquid nitrogen, in part with the gas phase.

For seeding into a bioreactor with mechanical agitation, high density cryopreserved cells were removed from the storage with liquid nitrogen, thawed and used for seeding in a closed 3-liter glass bioreactor (DASGIP; Jlich, Germany). Four or five tubes were removed from the storage with liquid nitrogen, the part with the gas phase, and placed directly in a 37 ° C water bath for 105 seconds. The contents of the thawed tubes were then transferred using a 2 ml glass pipette into a 50 ml conical tube. Then, 9 ml of medium (IH3 or Essential8 ™ (E8 ™)) containing 0.5% BSA and supplemented with 10 μmol / l of Rho-kinase inhibitor, Y-27632, was added dropwise to the tube. Then the cells were centrifuged at 80-200 OCS for 5 minutes. The supernatant from the tube was aspirated, 10 ml of fresh medium (IH3 or E8 ™) containing 0.5% BSA and supplemented with 10 µmol / l Rho-kinase inhibitor, Y-27632, was added, and the volume containing the cells was pipetted into transfer vials Wednesday (Cap2V8®, SaniSure, Murpark, California, USA). The contents of the vials were then injected directly into the bioreactor through sterile welded C-Flex tubes using a peristaltic pump. In preparation for planting pluripotent stem cells in a bioreactor, 1.5 l of medium was prepared (IH3 or E8 ™ with the addition of 0.5% BSA containing 10 μmol / l of Rho-kinase inhibitor, Y-27632), heated to 37 ° C, mixed at 70 revolutions per minute, adjusted to 6.8-7.1 pH using CO ₂ , with a level of dissolved oxygen of 30% (content of CO ₂ , air, O ₂ and N _{2 is} regulated). Immediately after seeding into the bioreactor, samples were taken from it to count the cells, and the average volume was adjusted as necessary to obtain a final cell concentration of 0.225 × 10 ⁶ cells / ml.

Cells seeded into a mechanically agitated bioreactor formed clusters of cells in a continuously stirred tank, and these clusters were kept in a pluripotency medium (IH3 or E8 ™, supplemented with 0.5% BSA) in the reactor for three days. The medium was changed daily, a partial replacement of the medium was performed 24 hours after seeding, 1-1.3 liters of the spent medium was removed and 1.5 liters of fresh medium was added. Forty-eight hours after sowing, 1.5-1.8 l of the spent medium was removed and 1.5 l of fresh medium was added. 72 hours after seeding, differentiation of pluripotent cells was initiated by removing> 90% of the medium used and adding differentiation medium (Table 7).

After the start of the process of differentiation, the cells were maintained for 12 or more days in a closed sterile suspension in a bioreactor with controlled temperature (37 °), pH 7.4 (for differentiation) with an established level of dissolved oxygen (10% DO at stage 1 and 30% DO at other stages, the content of CO2, O2, N2 and air is regulated). Throughout the entire differentiation process, with each medium change, the impeller was stopped for 5–20 minutes before the medium was removed through a dip tube to stabilize the clusters. The bioreactor medium was removed or added from / to a closed vial or bag using a peristaltic pump through a submerged tube connected to C-Flex® tubes using a Terumo ™ tube welder to maintain a closed system. The impeller and heater were restarted after adding sufficient medium to the vessel to immerse the wheel completely.

In order to control the process of the bioreactor, medium samples containing the cell clusters were collected daily to determine cell number and viability (NucleoCounter ^®), as shown in FIG. 7. The overall growth of cells was observed in the process, the seeded number of 0.225 × 10 ⁶ viable cells / ml grew to an average number of 0.92 × 10 ⁶ viable cells / ml at stage 4 days 3. While maintaining a certain level of acidity (pH 7.0 -6.8) during seeding into the bioreactor and clustering of iPS cells, the average cell yield at the 4-day stage 3 increased on average by 1.3 × 10 ⁶ cells / ml (Fig.7),

In addition to daily counts, the bioreactor media samples were analyzed by NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, Massachusetts, USA). It was noted that, according to reactor presets, the pH of stage 0 was acidic relative to the homeostatic standard pH 7.4, common to most nutrient media, and the pH of the medium in the reactor decreased during stage 0 as a result of cellular metabolism (FIG. . eight). These results correlate with the tendency to increase the concentration of lactic acid and decrease the level of glucose until the end of 6 days of differentiation (Figures 9 and 10). Together, these data suggest that the cells in the reactor grow rapidly and absorb glucose during stage 0 and the first two stages of differentiation (1-6 days). However, from the beginning of stage 3 onwards, cellular metabolism (decreased lactate levels and increased glucose levels) in the reactor does not correlate with a peak in the number of cells in stage 3, followed by a mismatch in cell density during the 4th stage.

It was necessary to determine whether the changes in pH and metabolism specific to the stage correspond to changes in mRNA expression patterns. Testing of cell samples from the bioreactor was performed using four Biosystems® Low Density Arrays arrays (Life Technologies Corporation, Carlsbad, California, USA) designated “pluripotency”, “definitive endoderm (DE)”, “intestinal tube” (GT) and "Stage 4 (S4)", after which the results were compared with reference data obtained from samples of undifferentiated H1 cell line (WB0106) hES as a control, in order to standardize the expression of all runs and arrays.

Also, for each stage of differentiation, gene expression was determined using the same arrays. Also observed was the fact that the seeding cell material thawed in the bioreactor showed undifferentiated patterns of gene expression at the stage 0 day 1 and stage 3, 0 day (24 and 72 hours after seeding in the bioreactor: Fig. 11, 12, 13 and 14) . These results correlate well with flow cytometry, which showed high levels of expression of CD9, SSEA4, TRA-1-60, TRA- and 1-81, as well as the absence of CXCR4 / CD184 (Fig. 15 and Table 8). Although flow cytometry and qRT-PCR gene expression analyzes showed robust and stable patterns of expression of pluripotent genes (CD9, NANOG, POU5F1, SOX2, TDGF, and ZFP42) in accordance with a stable pluripotent state, a moderate but unstable increase in gene expression was also observed for GATA, but the GATA was unchanged. RCU, MIXL1 and T; and ≥ 100-fold increase in expression of CER1, FGF17, FGF4 and GATA2 in some samples during stage 0 prior to directional differentiation (Figs. 16 and 17).

At the end of stage 0 (72 hours after seeding the reactor), the cells were transferred to differentiation medium (Table 7) containing MCX and GDF8. Twenty-four hours after this replacement of the medium, significant changes in the structure of gene expression were observed (Fig. 18 and 19), such as a ~ 700x increase in Foxa2 expression and a 1000x increase in CER1, EOMES, FGF17, FGF4, GATA4, GATA6, GCG, MIXL1 and T. These elevated levels of expression indicated that the cells had passed the mesodermal stage. It was also noted that CDX2 levels were increased at stage 1 day 1 compared to undifferentiated cells (470x increase in expression compared to control), however, this transient increase in expression level and CDX2 expression fell by 94% between stage 1, day 1 and stage 1, day 3, returning to a level comparable to that which was observed before the start of differentiation (Fig. 14, 19 and 21).

72 hours after exposure to the DE differentiation environment, the cells expressed a profile according to the specifications of the definitive endoderm, and peak levels of CXCR4 and Foxa2 and SOX17 were expressed at> 1000x relative to reference levels. In accordance with the parameters of the definitive endoderm, it was also noted that the expression of the CER1, EOMES, FGF17, FGF4, GATA4, GATA6, MKG MIXL1 and T GHG genes decreased from elevated levels observed at stage 1 for 1 day (Fig. 20 and 21).

Changes in gene expression observed by Qrt-PCR correlate with results obtained by flow cytometry. There was an almost complete transition from the CD9-expressing / CXCR4-negative population of pluripotent cells when differentiation was initiated (Fig. 15) to a homogeneous population of cells expressing CXCR4 (98.3% CXCR4-positive cells, ± 1.9 SD) at the end of stage 1 ( Fig. 22).

After the formation of the definitive endoderm (stage 1), the medium was replaced with another containing FGF7 morphogen, which is used to cause the formation of a primitive anterior intestine (stage 2). In accordance with the formation of the primitive gut, the levels of expression of HNF4α and GATA6 at stage 2, days 1 and 3, increase, while genes with a high level of expression at day 3 of stage 1 (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX17 and PV) showed a decrease in expression at the end of stage 2 (Fig. 23). Expression of the genes of the foregut (AFP, Pdx1 and Prox1) increased (Fig. 24).

After the cells were cultured in medium during stage 2 for 72 hours, the culture was transferred to stage 3 medium (Table 7). In this medium, cell expression markers correspond to the endoderm of the pancreatic line, one hundred times determined from the expression of PDX1 and FOXA2 (90.9% ± 11.9 CO of PDX1 positive and 99.2% ± 0.6 CO of FOXA2 positive), shown in FIG. 25. These results were confirmed by data from samples analyzed for gene expression using Qrt-PCR. The expression of the PDX1 genes increased 5-fold in 24 hours from the end of stage 2 day 3 (38,000-fold relative to H1) to the end of stage 3 day 1 (200,000-fold relative to H1) and again twice in 48 hours at stage 3 day 3 ( 435 000 times H1). These data indicated the formation of pancreatic cells (Fig. 26). This observation was further confirmed by an increased level of genes usually expressing in the pancreas (ARX, GAST, GCG, INS, Isl1, NEUROD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, PAX6, Ptf1a and SST), as shown in FIG. . 26. In addition, very low or absent OCT4 / POU5F1 expression was observed (2-10% relative to control or 32-37 Cts samples according to Qrt-PCR) and a high expression level of other markers of endodermal AFP, ALB, and CDX2 lines, which indicates on the specification and transition of the cell population in the bioreactor from the cells of the intestinal tube to the pancreatic cells

As shown in FIG. 27, at the end of the differentiation process at stage 4, day 3, cells maintain high levels of expression of PDX1 and FOXA2, and further, an expression pattern appears corresponding to a combination of pancreatic endocrine cells (28.1% ± 12.5 CO of chromogranin-positive) and precursor cells of pancreatic cells (58.3% ± 9.7 WITH NKX6.1-positive). Patterns of marker expression characteristic for this stage indicate an effective stepwise differentiation from the population of pluripotent cells into progenitor cells of pancreatic cells. The results obtained using flow cytometry were further confirmed by Qrt-PCR data. All genes commonly expressed in the pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST) showed an increased level of expression. (Fig. 28).

The expression pattern that is observed in FIG. 27, remained relevant during several runs with several process variations, for example, with different inoculum, stage 0 medium, pH of stage 0 medium and the use of an antifoam agent. Different sources of inoculum were tested and each time there was an effective generation of endodermal pancreatic cells with> 90% FOXA2,> 75% PDX1, and> 50% NKX6.1 (Fig. 29). In addition, there were no significant differences in the expression patterns of the product of the bioreactor between cells that were grown in stage 0 in a laboratory-developed medium called “IH3” with 0.5% BSA or commercially available medium: Essential8 ™ with 0.5 % BSA (Fig. 30). When investigating the role of pH in stage 0 culture, it was noted that cells grown in stage 0 with a relatively low pH (6.8) showed an increase in growth in the bioreactor relative to the average perspective (Fig.7), but no significant changes in cell profile on day 3 of stage 4 (Fig. 31). In addition, the use of an emulsifier C emulsion (Sigma catalog number A8011) in the amount of 94 parts per million clearly reduced the number of bubbles formed during the bubbling, but did not affect the cell profile from the end of stage 0 to the 4th stage of the day 3 cells (Table 9 and Fig. 32).

At the end of each differentiation, the cell product was cryoconserved in the bioreactor. Cells were washed in MCDB131 with 3.63 g / l sodium bicarbonate or MCDB131 with 3.63 g / l sodium bicarbonate, glucose (up to 8 mm) and 1 × Glutamax, and then transferred to cold (<4 ° C) medium for cryopreservation consisting of 57.5% MCDB131 with 2.43 g / l sodium bicarbonate, 30% xenobiotic-free KSR, 10% DMSO, and 2.5% HEPES (final concentration 25 mM). The cells were then frozen in a controlled-speed freezer (CRF) using a cooling profile that maintained cell clusters in a cryopreservation medium at ambient temperature for a maximum of 15 minutes, followed by a decrease in temperature to 4 ° C for 45 minutes and a further decrease in temperature at 2 ° C / min to -7 ° C (sample). Then the sample was rapidly cooled, the temperature in the chamber went down at a rate of 25 ° C / min to -45 ° C. Then it was planned to increase the temperature in the chamber by 10 ° C / min to -25 ° C (chamber). Then the sample was cooled at a rate of 0.2 ° C / min until the temperature reached -40 ° C. Then the camera was cooled to -160 ° C at a rate of 35 ° C / min and kept at this temperature for 15 minutes. Samples were transferred to a storage container with liquid nitrogen, part with a gas phase, for storage after termination of the CRF run.

Cells can be thawed by removing from the gas phase storage with liquid nitrogen and transferred to the vial in a water bath at 37 ° C. The tube was gently shaken in a water bath for less than 2 minutes until a small crystal of ice remained in the vial. The contents of the vessel were then transferred to a 50 ml conical flask and diluted dropwise over two minutes using MCDB131 medium with 2.43 g / l sodium bicarbonate and 2% BSA, to a final volume of 20 ml. The total number of cells was determined using a NucleoCounter ^® apparatus and the cell suspension was transferred to ultra small carriers in a culture dish for 1 hour. Then the cells were isolated from the medium in a 50 ml conical flask, the supernatant was removed and the cells were resuspended in medium 4 stage media for analysis or in vivo studies.

Alternatively, after defrosting, the cells from the vial were transferred to an empty 125 ml Corning® glass centrifuge tube (Corning, Incorporated, Corning, New York, USA) and 10 ml of MCDB131 medium containing 2.43 g / l Sodium bicarbonate and 2% BSA. The final volume was adjusted to 80 ml with the same medium. The total number of cells was determined using NucleoCounter ^® and the cell suspension was stirred at 40-65 revolutions per minute overnight (12-28 hours). The cells were then characterized or used for the i n vivo assay .

The composition of the medium IH3 is given below and is also described in US Patent Application Publication No. 2013/0236973, the disclosure of which is incorporated by reference in its entirety as it relates to a suitable culture medium. The amount of BSA in IH3 may vary.

The composition of the medium IH3 Base environment Additives DM-F12 1 × ITS-X
0.5% FAF-BSA reagent grade
1 ng / ml TGF-β1
100 ng / ml bFGF
20 ng / ml IGF-1
0.25 mM ascorbic acid.

Table 7 Start
Day / Date Stage 1 Stage 2 Stage 3 Stage 4 Base environment MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) Supplements 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 Growth factors Only day 1 and 2:
GDF8
100 ng / ml FGF7
50 ng / ml FGF7
50 ng / ml not Small molecules Only day 1:
MCX
[2 μm] RA [2 μM]
SANT [0.25 μM]
TPPB [100 nM]
Only day 1
LDN 100 SANT [0.25 μM]
TPPB [100 nM] Day 3 3 3 3 NOTES :
All days relative to 0 h. Medium change
Day 1 and 2,
No replacement for day 3 Medium change
Day 1 and 3,
No replacement for day 2 Medium change
Day 1 and 2,
No replacement for day 3 In the case of S4 growth, replace the medium on day 1 and by the end of day 3

Table 8 BX Replication Seed material CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 one KC 83.3 0.1 99.9 94.5 85,8 2 Hw 95.5 0.2 100 91 84 3 ISM (pink) 95,8 0.1 100 76.1 36.5 four ISM (pink) 93.2 0 99.9 78.6 64.5 five ISM 1 97,8 0.2 99 74.8 66.4 6 ISM 2 98.6 0.2 100 92.2 86 7 ISM 1 98.1 0.1 99.9 88,8 80.3 eight ISM 1 99.1 0.1 99.9 93,8 83.3 9 ISM 2 97.2 0.1 99.9 88.3 81 ten ISM5 98 0.1 99.3 93.1 85.7 eleven ISM6 72.6 0.2 99.9 94.7 88.9 12 ISM6 85.9 0.7 99.4 71.9 54.1 CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 The average 93.6 0.1 99.8 87,8 76.6 Art. deviation 8.3 0.1 0.3 7,6 15.5

Table 9 Stage-day-time The density of viable cells (million cells / ml) CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 S0D3-24H 0.626 95,8 0.1 99.8 87.9 74 The density of viable cells (million cells / ml) CD9 CD184 CD99 S1D3-24H 0.9 50.7 98.9 99 The density of viable cells (million cells / ml) NKX6.1 Khoromatogranin NKX2.2 PDX1 FOXA2 S4D1-24H 0.943 69.3 14.2 23.6 98.8 99.7 The density of viable cells (million cells / ml) NKX6.1 Khoromatogranin CDX2 SOX2 NKX2.2 PDX1 FOXA2 NEUROD S4D3-24H 1,002 66.2 35.6 0.3 15.8 38.1 99 99 45.6

Materials:

• Cells of the human embryonic cell line H1 (hES), (cells WA01, WiCell, Madison WI)

• PBS (Cat. No. 14190, Invitrogen)

• Y-27632 (Axxora Part No. ALX-270-333, San Diego, CA)

• EDTA, (Lonza, catalog number 17-7-11E)

• NucleoCounter®- (ChemoMetec A / S, catalog number YC-T100, Allerod, Denmark)

• Processed 6-well plates with two non-woven cultures (Becton Dickinson, Catalog No. Falcon 351146, Franklin Lakes, NJ)

• Accutase®, (Sigma, catalog number A-6964, St. Louis, Missouri, USA)

• Bioreactor sensors for determining pH and dissolved oxygen (DO) (FermProbe ^® pH electrode 225 mm, model number F-635, OxyProbe ^® dissolved oxygen sensor 12 mm, model number D-145 from Broadley-James Corporation, Irwin state of California, USA)

• An immuno-protective macroencapsulation device (TheraCyte ™, Irvine, California, USA)

• mM ELISA of human C-peptide (MERCODIA catalog number 10-1141-01)

• GlutaMAX ™, MCDB131 and Invitrogen ITS-X

• FAF-BSA (Proliant)

• Retinoic acid, glucose 45% (2.5M), SANT (Shh inhibitor) (Sigma)

• GDF8 (Peprotech)

• MCX

• FGF7 (R & D Systems)

• LDN-193189 (BMP receptor antagonist) (Stemgent)

• TPPB (PKC activator) (ChemPartner)

• Custom Made Environment MCDB 131

Example 8

Maturation and function obtained in the bioreactor and cryopreserved clusters of pancreatic precursor cells

To generate a sufficient number of cells for each, bioreactors thawed one vial of H1 hES (WB0106) cell line 31 passages from the main cell bank. The cells were grown adherent in mTeSR ^® 1 for several passages on Matrigel ™ using EDTA passaging until a sufficient number of cells were obtained to sow five Matrigel ™ coated double layer cells for CellSTACKs® (CS2). The cells were grown attached and, after forming a confluent layer in CS2 by 70%, a system of tubes with C-Flex® caps was attached to the medium ports, to which tubes leading to the pump were connected to close the system. After the system was closed using C-Flex® tubes, bags or bottles were welded to it using the Terumo machine, where fluid volumes (medium, PBS ^{- / -} , Accutase ^®, or suspended cells) were transferred with a peristaltic pump.

For separation of CS2 cells, the cells were washed once with PBS Dulbecco without calcium or magnesium (PBS ^{- / -),} then treated with a solution of half Accutase ^® efficiency diluted PBS ^{- /} and incubated for 4-5 minutes. Accutase ^® solution was then removed, and after 3 minutes after application of the enzyme solution was added CS2 to stimulate cellular compartment. A container with mTeSR ^® 1 medium containing 10 μmol / l of Rho-kinase inhibitor, Y-27632, was pumped into CS2 to wash and inactivate residual Accutase ^® , then the wash was collected. A second wash volume was added, the wash was collected and combined with the first. 1.6-2.0 × 10 ⁹ cells were recovered from CS2s in a final volume of 2 liters. 2.0-2.5 × 10 ⁸ cells per layer were transferred to four CS2 or eight Cell Stacks ™ single-layer containers and incubated at 37 ° C for 2 hours in a humidified incubator with 5% CO ₂ in a volume of 200 ml per layer.

Using a closed loop C-Flex® tube with an attached tube for a pump attached between the ports of the CellSTACK® medium, the cell suspension was ground for 5 minutes at 75 revolutions per minute using a peristaltic pump to homogenize the aggregates. CellSTACKs® were then incubated overnight at 37 ° C for 18 hours in a humidified 5% CO ₂ incubator. 2 liters of cells and media from CellStack ™ were then combined and transferred, 1 liter each, into two 3 liters DASGIP bioreactor, together with 1.5 liters of fresh mTeSR® medium per bioreactor. Cells were maintained for two additional days in the mTeSR® medium prior to differentiation, with complete replacement of the medium 24 hours after seeding the bioreactor. After that, differentiation was initiated and directed, as described in Table 10. The cells were maintained for a total of 14 or 15 days (2 days mTeSR® + 12 or 13 days in the differentiation process) in a closed sterile suspension in a controlled temperature bioreactor (37 ° C) , pH (adjustable or regulated CO ₂ to 6.8 or 7.2 for pluripotent cells and 7.4 for differentiation) and the level of dissolved oxygen (30% DO, CO ₂ / air levels are regulated). The impeller was stopped for 5-20 minutes before removing the medium through the immersion tube to stabilize the clusters. Medium was removed or added using a peristaltic pump through a submerged tube connected to C-Flex® tubes (Cole-Parmer North America, Vernon Hills, Illinois, USA) using a Terumo ™ tube welder to maintain a closed system. The impeller and heater were restarted after adding enough medium to completely immerse the wheel.

Two production runs were started in 3 liter reactors using these methods. In the first run of the reactor, two different pH values were tested during the first two days in a pluripotent culture medium. Reactor 1 was set to a pH of 7.2 with a fixed infusion rate of CO ₂ gas _of 5%, so that the pH decreased as the medium of the reactor oxidized over time due to metabolic activity of the cells. Reactor 2 was set to a pH of 7.2 with an adjustable level of CO ₂ gas. In the second run of the reactor, the pH was set at 6.8 for reactor 1 and 7.2 for reactor 2 in both regulated gas levels of CO ₂ .

In order to control the processes in the bioreactor, at the end of each stage of differentiation, samples of cell clusters were taken and analyzed using flow cytometry (table 11; table 12). There was an almost complete transition from the CD9-expressing / CXCR4-negative population of pluripotent cells when initiating differentiation to a homogeneous population of cells expressing CXCR4 (96.9-98.1% of CXCR4-positive cells) upon completion of the formation of definitive endoderm.

The results obtained using flow cytometry correlate with the results of paired samples analyzed by RT-PCR. Samples were tested throughout the process to evaluate the characteristics of gene expression during differentiation from pluripotency to pancreatic transformation. Prior to the start of directed differentiation, mRNA of cell clusters in the bioreactor was tested on Low Density arrays for a panel of genes associated with pluripotency or early onset of differentiation.

It was noted that cells from the bioreactor retain gene expression characteristic of pluripotency (POU5F1, NANOG, Sox2 and ZFP42) and show minimal or absent induction of genes characteristic of differentiation (AFP and Foxa2: <50-fold increase; FOXD3, GATA2, GATA4 , GSC, HAND2, MIXL1 and T: <10-fold increase in expression) compared with control undifferentiated H1 cell line (Fig. 33). However, as soon as the cells came into contact with the differentiation medium at stage 1 day 1, the expression patterns changed dramatically, the expression levels of CDX2, CER1, FGF17, FGF4, Foxa2, GATA4, GATA6, MCHX1, MCHX6, and Brachyury (T) increased to 100 -1000 times relative to the levels of undifferentiated cells of the HES H1 line (Fig.34). By the end of stage 1, day 3 (formation of definitive endoderm), expression of CD9, CDX2, FGF4, MIXL1, NANOG, POU5F1 and Brachyury (T) decreased relative to that of stage 1 day 1, and expression of characteristic final endoderm genes such as CD99, CER1 , CXCR4, FGF17, GATA4, GATA6, KIT, OTX or SOX17, reached a maximum (Fig. 35).

At the end of stage 1, the cell culture medium was replaced: instead of the medium containing GDF8, the medium containing FGF7 was added. Several different gene expression patterns were noted: increased expression of certain genes during stage 2 (AFP, Atoh1, HHEX, OSR1, Pdx1, Prox1, Sox2 and SOX9), reduced expression (Hand1 and SOX17), consistently high constant expression (HNF4α), or low / missing expression (CDX2, GAST, Nkx2.2, Nkx6.1 and Ptf1a) (Fig. 36a-e). These models showed that the cells in the reactor acquire the characteristics of the cells of the foregut (AFP, Atoh1, HHEX, HNF4α, OSR1, Pdx1, Prox1, Sox2 and SOX9), the expression of mesoderm markers (Hand1 and SOX17) decreased. However, by the end of stage 2, cells were not yet more specifically specialized in mature intestinal cells or pancreatic cells (CDX2, GAST, Nkx2.2, Nkx6.1 and Ptf1a).

At the end of stage 3, cells were specialized in pancreatic, as determined by the expression of Pdx1, which showed a> 100,000-fold increase in mRNA relative to the undifferentiated control (Fig. 36) and the results of flow cytometry, which showed 76-98% of cells expressing Pdx1 (Table 11) . Induction of other pancreatic genes (GAST, NKX2.2, NKX6.1, PROX1, PTF1a and SOX9) and intestines, such as AFP and CDX2, was also observed; which indicated gradual maturation and cell specification.

By the end of the differentiation process on day 3 or 4 of stage 4, the cells showed expression patterns corresponding to a combination of pancreatic endocrine cells (47-54% chromagranin-positive) and pancreatic progenitor cells (33-52% NKX6.1-positive), as shown in tables 11 and 12. These patterns are stage-specific expression of markers indicate the effective stepwise differentiation of pluripotent cell populations in the pancreatic progenitor cells are characterized by high level expression of Pdx1 (> 1 × ¹⁰ June cr full induction) and other pancreatic genes (> 1000 times induction of ARX, GCG, GAST, INS, ISL, NEUROD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Ptf1a, and SST) and an almost total loss of OCT4 / POU5F1 expression compared to undifferentiated human embryonic stem cells of the H1 human line (Fig. 37).

At the end of the differentiation process, 0.08-0.45 × 10 ⁶ cells / ml was obtained (FIG. 38: daily count of the number of cells). The cells obtained in this process were cryopreserved or directly implanted in animals either subcutaneously using a TheraCyte ™ device or under a renal capsule. For cryopreservation of cells, they were transferred to cryopreservation medium consisting of 57.5% MCDB131 with 2.43 g / l of sodium bicarbonate, 30% xenobiotic-free KSR, 10% DMSO and 2.5% HEPES (final concentration 25 mM). After the cell clusters were suspended in a cryopreservation medium at room temperature, the cryopreservation tubes were transferred to a freezer at a controlled rate (CRF) for 15 minutes. The temperature in the chamber was then lowered to 4 ° C over 45 minutes, and further lowered by 2 ° C / min to -7 ° C (sample). Then the sample was rapidly cooled, the temperature in the chamber went down at a rate of 25 ° C / min to -45 ° C. Then it was planned to increase the temperature in the chamber by 10 ° C / min to -25 ° C (chamber). Then the sample was cooled at a rate of 0.2 ° C / min until the temperature reached -40 ° C. Then the camera was cooled to -160 ° C at a rate of 35 ° C / min and kept at this temperature for 15 minutes. Samples were transferred to a storage container with liquid nitrogen, part with a gas phase, for storage after termination of the CRF run.

After the cells were stored in the gas phase of liquid nitrogen, they were thawed by removing from storage and transferring to a water bath at 37 ° C. The tube was gently shaken in a water bath for less than 2 minutes until a small crystal of ice remained in the vial. The contents of the vessel were then transferred to a 50 ml conical flask and diluted dropwise over two minutes using MCDB131 medium with 2.43 g / l sodium bicarbonate and 2% BSA, to a final volume of 20 ml. The total number of cells was determined using a NucleoCounter ^® apparatus and the cell suspension was transferred to ultra small carriers in a culture dish for 1 hour. The cells were then isolated from the medium in a 50 ml conical flask, the supernatant was removed, and the cells were resuspended in 4 stages of medium. The cells were then implanted in animals either subcutaneously using a TheraCyte ™ device, or under a renal capsule, or the cells were incubated in Petri dishes with ultra-low binding capacity overnight and then implanted into an animal.

The animals tracked the level of glucose and C-peptide in the blood every four weeks after implantation of the graft. Animals implanted with non-cryopreserved pancreatic progenitor cells using the TheraCyte ™ device or by directly placing the cells under the renal capsule showed cell maturation and C-peptide expression 1 ng / ml after 16 weeks, later rising to 2 ng / ml C peptide 20 weeks after implantation (Fig. 39a and 39d). In addition, when treating STZ for ablation of β-cell function, animals with implants maintained normoglycemia prior to graft removal, thereby indicating that the grafts were able to protect animals from diabetes caused by a single large dose of STZ (Fig. 39b).

This pattern was observed in animals that were implanted with cryopreserved cells. Animals that were implanted with cryopreserved pancreatic progenitor cells under the renal capsule, which were cultured for 1 hour after thawing (1207B), showed an average amount of C-peptide of 0.56 ng / ml and 1.09 ng / ml after 16 and 20 weeks respectively, while cells cultured overnight after defrosting (120 ° C) showed an average of 0.81 ng / ml and 1.35 ng / ml of the C-peptide at 16 and 20 weeks, respectively (Fig. 39d). Animals that were implanted with cryopreserved pancreatic progenitor cells using the TheraCyte ™ device showed more than 1 ng / ml C-peptide after 16 weeks, and also as non-cryopreserved controls, were able to express therapeutic levels of C-peptide a week after treatment STZ (0.98 ng / ml, Fig. 39c). These results indicate that pancreatic cryopreserved progenitor cells can function with an efficacy comparable to that of non-cryopreserved controls when tested in animal models.

Table 10 Original day / date Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Base environment mTeSR®1 MCDB131 (3.64 g / l NaCO ₃ ) MCDB131
(3.64 g / l NaCO ₃ ) MCDB131
(3.64 g / l NaCO ₃ ) MCDB131
(3.64 g / l NaCO ₃ ) Supplements 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 Growth factors Only day 2:
GDF8
100 ng / ml FGF7
50 ng / ml FGF7
50 ng / ml not Small molecules Y-27632
(day 0 only)
[1: 1000; 10 μmol] Only day 1:
MCX
[3 μM] RA [2 μM]
SANT [0.25 μM]
TPPB [100 nM]
Only day 1
LDN 100 SANT [0.25 μM]
TPPB [100 nM] Day 3 3 3 3 3 NOTES : Medium change
Day 1 and 2,
No replacement for c3 Medium change
Day 1 and 3,
No replacement for day 2 Medium change
Day 1 and 2,
No replacement for c3 Replacing the environment only per day 1
Glucose bolus on day 3

Note:

• The base medium in this table 10 may optionally contain 5 mM glucose in steps 1-5, provided that Glutamax is not used as an additive.

• Table 10 above also indicates that ([100 nM]) Cypi can be added in step 4.

Table 11 Pluripotency Day process Title CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 2 Bx1 78.9 0.1 100 54.5 51.1 Bx2 66.5 0.0 100 63.5 72.3 Title CD9 CD184 DE (St1sut2) four Bx1 9.9 87.9 Bx2 19.7 83.1 DE (St1sut3) five Bx1 17.4 98.1 Bx2 25.4 96.9 Title Nkx6.1 Nkx2.2 PDX1 PE (St3Sut3) eleven Bx1 4.4 25.2 98.6 Bx2 4.8 28.9 76.2 Title Nkx6.1 Synaptophysin CDX2 Sox2 Nkx2.2 Chromogranin PPC (St4Sut3) 14 Bx1 33.2 67.4 2.1 13.0 69.3 51.1 Bx2 35.1 56.9 1.9 11.5 64.4 51.2

Table 12 Pluripotency Day process Title CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 2 Bx1 99.8 0.3 100.0 88.6 85,8 Bx2 99.8 0.3 100.0 86,8 85.9 Title CD9 CD184 CD99 DE (St1sut3) five Bx1 88.3 99.2 97.0 Bx2 78.3 99.3 96.9 Title Nkx6.1 Nkx2.2 Chromogranin PE (St3Sut3) eleven Bx1 6.3 23.2 8.5 Bx2 1.2 24.6 11.5 Title Nkx6.1 Synaptophysin CDX2 Sox2 Nkx2.2 Chromogranin PPC (St4Sut3) 14 Bx1 49.0 7.3 13.1 56.1 49.2 Bx2 52.6 3.1 19.9 54.5 47.4 PPC (St4Sut4) 15 Bx1 48.4 53.1 0.4 4.9 60.3 44.3 Bx2 45.7 66.5 0.2 4.5 63.7 54.3

Calculation of shear stress experienced by cell aggregates in a bioreactor with mechanical agitation

A shear stress was determined that cell aggregates of 2.7 liters of the stirred DASGIP suspension in the bioreactor were tested at a mixing speed of 70 rpm in a 3l DASGIP bioreactor. In order to calculate the shear stress values, the following stated assumptions were made.

Assumptions:

1. The maximum shear stress experienced by cell aggregates is not the result of turbulent eddies.

2. The maximum shear stress experienced by cell aggregates is not the result of the friction of the two aggregates against each other or the aggregate against the blade.

3. Voltage. caused by collisions with partitions (i.e. immersed tubes and probes) are not considered in these calculations.

For the purposes of calculations in this description, the nomenclature and physical parameters listed below were used.

Nomenclature:

Reduction Units

Fluid density kg / m ³

Fluid viscosity Pass

Kinematic viscosity m ² / s

Maximum shear stress dyn / cm ² N Stirring rev / sec P Electricity consumption kg m ² / s ³ P _n Power number without units Re Reynolds number without units

Power dissipated per mass unit m ² / s ³ D _i Blade wheel diameter M D Diameter of tank M W Paddle Width M V _l Fluid volume m ³ K ₁ -K ₄ Calculated values based on empirical correlations NAGATA

Options :

Bioreactor parameters D _i 0.08 m D 0.13 m W 0.04 m V _l 0,0024 m3

Environment settings

Density (

1000 kg / m ³ Viscosity (

8.50E-04 Pass kinematic viscosity (

8.50E-07 m ² / s

The listed parameters of the bioreactor and the medium were used in the equations below.

Equations :

Reynolds numbers:

Maximum shear stress on aggregates (Cherry and Kwon 1990)

Power dissipated (ε) per unit mass

Consumed electricity (P)

The calculation of the power number was based on the empirical correlation obtained by Nagata (1975) for containers without obstacles with mechanical agitation.

Where

The maximum shift of cell aggregates was calculated at a stirring speed of 70 revolutions per minute in a 2.7 L DASGIP bioreactor; it was at least 0.25 pascals (2.5 dyn / cm ² ). Cells contained in the outer layer of the clusters experienced the highest levels of shear stress. These shear stress values are highly dependent on the assumptions made.

Example 9

Differentiation of embryonic stem cells of the WA01 cell line into definitive endoderm cells: the role of MCX / GDF8 in suspension culture

Clusters of pluripotent human embryonic stem cells from the H1 stem cell line (NIH code: WA01) were seeded at a cell density ranging from 0.25 × 10 ⁶ to 2 × 10 ⁶ cells / ml in Erlenmeyer flasks / stirred flasks, centrifuge tubes or on uncovered Low-binding 6-well plates or non-tissue culture-treated 6-well plates are seeded on MCDB-131 medium containing 3.64 g / ml sodium bicarbonate and 5.5 mM glucose (Catalog No. A13051 DJ, Invitrogen, CA), supplemented with 2% fatty acid-purified BSA (Catalog No. 68700, Proliant, I A), 1X GlutaMAX ™ (Catalog No. 35050-079, Invitrogen, Calif.), Additionally added 2.5 mM glucose (Catalog No. G8769, Sigma) and ITS-x with a concentration of 1: 50,000 base solution (Catalog No. 51500056, Invitrogen, California). MCDB-131, as supplemented in this way, will be referred to as the “Stage 1 Baseline Environment” in this document. Clusters in this medium on the first day of differentiation were treated with either 3 μM MCX (GSK3B inhibitor, 14-prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ .1 ~ 8.12 ~] heptacosis-1 (25), 2 (27), 3.5.8 (26), 9,11,21,23-nonen-16-one, patent application US No. 12 / 494,789; incorporated herein by reference in full) and 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), or only 3 μM MCX, or 20 ng / ml WNT-3A ( Catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml of activin A (Catalog No. 338-AC, R & D Systems, Minnesota, USA), or 20 ng / ml WNT-3A only . On the second day, the cells were transferred to a fresh base medium of stage 1 with the addition of either 100 ng / ml GDF8, or 100 ng / ml activin A. Samples were collected for flow cytometry, PCR and Western blot analysis at various times, starting from zero time (immediately before adding the base medium plus additives) until 72 hours after the start of differentiation.

The efficiency with which the definitive endoderm was generated was determined after 3 days of differentiation under each condition, measured using flow cytometry, of the percentage of cells expressing cell surface markers CXCR4, CD99 and CD9. The data (as shown in the expression graphs in Fig. 40a-d and summarized in Table 13) show that in suspension culture, adding 3 μM MCX in the absence of substances of the TGF-β family during the first days of differentiation generates definitive endoderm at levels comparable to those that appear by treating cells with 3 μM MCX plus 100 ng / ml GDF-8 or 20 ng / ml WNT-3A plus 100 ng / ml activin A in one day.

Table 13 Treatment
(Day 1 → Day 2 and 3) CD9
(% through FACS analysis) CD99
(% through FACS analysis) CD184
(% of parent) MCX + GDF8 → GDF8 1.5 0.0 95.3 / 95.4 only MCX → GDF8 6.4 0.0 93.6 / 93.6 WNT3a + activin A → activin A 3.3 22.1 98.1 / 97.5 Only WNT3a → Activin A 31.7 6.2 87.8 / 86.1

Example 10

Differentiation of embryonic stem cells from the WA01 cell line to the definitive endoderm: dose-dependent effect of MCX compounds in suspension culture depending on concentration

Clusters of pluripotent embryonic human stem cells from the H1 stem cell line (NIH code: WA01) were seeded at a cell density ranging from 0.25 × 10 ⁶ to 2 × 10 ⁶ cells / ml in Erlenmeyer flasks / stirred flasks, centrifuge tubes on base medium stages 1 as described in example 9. The clusters were treated with a base medium of stage 1 containing 1.5, 2, 3 or 4 μmol MCX on the first day of differentiation and fresh base medium stage 1 containing 100 ng / ml GDF-8 on 2 day. On the third day, Wednesday was replaced. Samples were collected for flow cytometry and PCR analysis after the third day of differentiation.

The efficiency with which the definitive endoderm was generated was determined by measurement using flow cytometry of the percentage of cells expressing cell surface markers CXCR4, CD99 and CD9. The data (as shown in the expression graphs in Fig. 41A-D and summarized in Table 14) show that in suspension cultures, the addition of MCX at concentrations of less than 2 μM results in a smaller number of definitive endoderm cells (as indicated by a lower percentage CXCR4 positive cells and a higher percentage of CD9 positive cells). In addition, at concentrations above 4 μM, MCX exerts a deleterious effect on the cells, which leads to a decrease in cell viability. However, by increasing the concentration of BSA, the effects of MCX can be modulated so that a concentration of ≥ 4 µM / L can be applied. On the other hand, concentrations of ≤ 1.5 μMol / L can be used to create definitive endoderm using lower BSA concentrations.

Table 14 Treatment CD9 (% by FACS) CD184 (% by FACS) 4 μM MCX 1.0 95.2 3 μM MCX 0.2 96.0 2 μM MCX 0.2 96.5 1.5 μM MCX 68.4 67,8

Example 11

Differentiation of embryonic stem cells from the WA01 cell line into the definitive endoderm: the role of the medium replacement rate in suspension culture

Clusters of pluripotent embryonic human stem cells from the H1 stem cell line (NIH code: WA01) were seeded at a cell density ranging from 0.25 × 10 ⁶ to 2 × 10 ⁶ cells / ml in Erlenmeyer flasks / stirred flasks, centrifuge tubes on base medium stages 1 as described in example 9. The clusters were treated with base medium of stage 1 containing 3 μM MCX on the first day of differentiation and fresh base medium of stage 1 containing 100 ng / ml GDF-8 on day 2. In control cultures, the medium was changed on the third day; in a separate vessel, the medium was not replaced on the third day. Samples were collected for flow cytometry and PCR analysis after the third day of differentiation.

The efficacy with which the definitive endoderm was generated was determined at each of the conditions by measuring, using flow cytometry, the percentage of cells expressing cell surface markers CXCR4, CD99 and CD9. Results are shown in expression graphs. FIG. 42A and B and are shown in Table 15.

Table 15 Treatment CD9 (% by FACS) CD99 (% by FACS) CD184 (% by FACS) Complete replacement of medium at stage 1 0.2 72.4 90.2 / 89.6 Replacement was not carried out at stage 1 day 3 0.9 68.3 89.2 / 89.8

Example 12

Differentiation of embryonic stem cells from the WA01 cell line to the definitive endoderm: Use of GlutaMAX ™ in suspension culture

Clusters of pluripotent embryonic human stem cells from the H1 stem cell line (NIH code: WA01) were seeded at a cell density ranging from 0.25 × 10 ⁶ to 2 × 10 ⁶ cells / ml in Erlenmeyer flasks / stirred flasks, centrifuge tubes.

This example was performed to determine if Glutamax ™ was required to generate definitive endoderm by suspending clusters in the base medium of stage 1 (as described in example 9) with and without GlutaMAX ™, the medium was supplemented with 3 μM MCX on the first day of differentiation with the replacement of the base medium of stage 1 containing 100 ng / ml GDF-8 for 2 days. On the third day, Wednesday was replaced. Samples were collected for flow cytometry and PCR analysis after the third day of differentiation.

The efficacy with which the definitive endoderm was generated was determined at each of the conditions by measuring, using flow cytometry, the percentage of cells expressing cell surface markers CXCR4, CD99 and CD9. The results are shown in the expression graphs in FIG. 43A and B and are shown in Table 16.

Table 16 Treatment CD9 (% by FACS) CD99 (% by FACS) CD184 (% by FACS) X GlutaMAX ™ 0.2 93.7 96.8 / 96.7 0 GlutaMAX ™ 1,3 95.6 97.7 / 97.3

Example 13

Differentiation of embryonic stem cells from the WA01 cell line to definitive endoderm: the role of sodium bicarbonate concentrations in suspension culture

Clusters of pluripotent embryonic human stem cells from the H1 stem cell line (NIH code: WA01) were seeded at a cell density ranging from 0.25 × 10⁶ up to 2 × 10⁶ cells / ml in Erlenmeyer flasks / stirred flasks, centrifuge tubes on base medium stage 1 as described in example 9 (medium contained 3.64 g / l sodium bicarbonate), or on modified base medium stage 1, which contained 2.43 g / l sodium bicarbonate. The clusters were treated with basic medium of stage 1, containing MCX and GDF-8, as described in example 12. Samples were collected for flow cytometry after the third day of differentiation. Phase-contrast images were also made every day of differentiation.

The efficiency with which the definitive endoderm was generated was determined by measurement using flow cytometry of the percentage of cells expressing cell surface markers CXCR4, CD99 and CD9. The results are shown in the expression graphs in FIG. 44a and B and are shown in Table 17. In suspension cultures, a low level of sodium bicarbonate, 2.43 g / l, appears to lead to less effective generation of definitive endoderm (on average, 87.4% of cells express CXCR4), in contrast to from a higher level of 3.64 g / l (on average, 97.35% of cells express CXCR4). In addition, it was noted that differences in bicarbonate levels correlated with differences in cluster morphology at the end of stage 1, which was noted using phase-contrast microscopy (Fig. 44 C and D). In addition, cells differentiated at high bicarbonate levels, as observed, formed less dense cell clusters than cells differentiated at 2.43 g / l bicarbonate.

Table 17 Treatment CD9 (% by FACS) CD99 (% by FACS) CD184 (% by FACS) 3.64 g / l sodium bicarbonate 5.5 92.7 97.7 / 97.0 2,43g / l sodium bicarbonate 12.3 66.7 86.4 / 88.4

Example 14

Creating clusters of pancreatic progenitor cells from human induced pluripotent stem cells in a scalable process in a bioreactor

Cell therapy requires a large number (> 10 ⁸ ) of cells per dose. This example demonstrates a process capable of differentiating the induced masses of pluripotent stem cells (IPS cells) in quantities that are 3-5 orders of magnitude larger than current production practices of cell therapy allow.

In this example, the IPS-UTC cell line was used (obtained from umbilical cord tissue cells previously described in US patent application no. 13 / 330,931 (US patent application publication No. 2013/0157365), the disclosure of which is incorporated by reference in its entirety. to obtain cell lines iPS). Cells were obtained from mouse embryonic feeder cells using plasmid transfection without leaving traces and cryopreserved upon passaging 15.

Of these cryopreserved cells, a series of cell banks was created by adding the contents of the starting material tube during the defrosting process of the vial to the recombinant human laminin (hrLaminin LN-521 catalog number from Biolamina, Stockholm, Sweden) in Essential8 ™ (E8) ™ medium Life Technologies Corporation (Grand Island, New York, USA) for the generation of laboratory seed. The thawed and grown material is called the “pre-pre-main cell bank” (Pre-Pre CEM) and serves as seed for future banks. Of the three consecutive cell banks, the Pre-Pre MCB then generates a provisional master cell bank (Pre MCB), a master cell bank (MCB), and a working cell bank (WCB). One vial of WCB is then thawed, grown on hrLaminin using EDTA passaging for three passages in an E8 ™. Cells were first seeded after defrosting into a T225 flask (Corning; Corning, New York, USA), and then dissected into several T225 flasks. Several T225 flasks were then passaged and pooled to inoculate a single monolayer cell stack cell stack (CS1). After the cells CS1 were combined, the cells were washed once with PBS ^{- / -,} treated with a solution of half Accutase ^® efficiency, diluted with PBS ^{- / -} and incubated for 4-5 minutes. Then Accutase ^® was removed and after 3 minutes after application of the enzyme solution, CS1 was used to stimulate cellular compartment. E8 ™ supplemented with 0.5% BSA containing 10 µmol / L Rho kinase inhibitor, Y-27632, was added to CS1 to wash and inactivate residual Accutase ^® . The flush was collected and a second flush volume was added, the flush from which was also collected and combined with the first flush.

Cells were transferred to medium supplemented with 0.5% BSA containing 10 μmol / l Rho kinase inhibitor, Y-27632, into a 1-liter disposable centrifuge tube (Corning Corning, NY, USA) at a concentration of 1 × 10 ⁶ cells / ml in 225 ml. The cells were allowed to cluster in a static suspension for 60 minutes in a humidified 5% CO2 incubator, then mixed for 5 minutes at 55-65 revolutions per minute and 225 ml of additional medium added, supplemented with 0.5% BSA containing 10 µmol / l inhibitor of Rho-kinase, Y-27632. The cells were allowed to settle in a static culture for 30 additional minutes, and then 150 ml of additional medium was added to the centrifuge tube with the addition of 0.5% BSA containing 10 μmol / l of Rho-kinase inhibitor, Y-27632. After that, the cells were continuously mixed at a speed of 50-70 revolutions per minute in a humidified incubator with 5% CO ₂ . Twenty-four hours later, the centrifuge tube was removed from the incubator and the clusters were allowed to settle for 5-10 minutes. Then the medium was aspirated until 200 ml remained and 400 ml of additional fresh culture medium was added to the centrifuge tube. This process was repeated at the end of 2 days (48 hours after transfer).

72 hours after the initial Accutase ^® treatment, the process of cell dissociation of clusters and seeding (passaging) in a centrifuge tube was repeated to keep the cells in suspension for repeated passaging (test range: 1-10 passages).

Using this process, pluripotent UTC cells were transformed from a culture attached to a substrate to a suspension culture as clusters of cells and then grown in suspension. These suspensions were subcultured and the cultured cells were then cryopreserved and stored for later use. In order to prepare a slurry overgrown cells for cryopreservation, cell clusters were dissociated with Accutase ^®, as described above, except that the cells were not passed through a cell strainer 40 micromolar. Cells in each disposable 1-liter flask were then counted, pooled as necessary, and centrifuged for 5 minutes at 80-200 OCS. The supernatant was then removed as completely as possible without disturbing the cell pellet. The cold (<4 ° C) CryoStor®10 was then added dropwise in such a way as to reach a final concentration of 150 million cells per ml, and the cell solution was kept in an ice bath while transferring to a 1.8 ml Corning Cryoconservation Vial (Corning; g Corning, New York, USA) or 15 ml package for cryoconservation Miltenyi (Miltenyi Biotec Inc. Auburn, California, USA).

The suspension of overgrown cells was then frozen in a vial at high density in a freezer at a controlled rate as follows. The chamber was pre-cooled to 4 ° C and the temperature was maintained until the test tube with the sample reached 6 ° C. Then the temperature in the chamber was lowered at a rate of 2 ° C / min until the test tube with the sample reached -7 ° C. After the sample tube reached -7 ° C, the chamber was cooled at 20 ° C / min to -45 ° C. After that, the temperature in the chamber was raised by 10 ° C / min until the temperature in the chamber reached -25 ° C, and then the chamber was further cooled at 0.8 ° C / min until the test tube with the sample reached temperatures of -45 ° C. The temperature in the chamber was then reduced by 35 ° C / min until the temperature in the chamber reached -160 ° C. The temperature in the chamber was then kept at -160 ° C for at least 10 minutes, after which the tubes were transferred to a storage facility with liquid nitrogen, in part with the gas phase.

For seeding in a bioreactor with mechanical agitation, high density cryopreserved cells were removed from the storage with liquid nitrogen, thawed and used for seeding in a closed 0.2-liter glass bioreactor (DASGIP; Jlich, Germany). Cryopreservation tubes were removed from the storage with liquid nitrogen, portions with a gas phase, and placed directly in a water bath at 37 ° C for 105 seconds. The contents of the thawed tubes were then transferred using a 2 ml glass pipette into a 50 ml conical tube. Then, 9 ml of E8 ™ containing 0.5% BSA with 10 μmol / L of Rho-kinase inhibitor, Y-27632, was added dropwise to the tube. Then the cells were centrifuged at 80-200 OCS for 5 minutes. After that, the supernatant was aspirated from the tube and 10 ml of fresh E8 containing 0.5% BSA and supplemented with 10 µmol / L of Rho-kinase inhibitor, Y-27632, were added. This volume containing cells was pipetted into vials for transferring medium (Cap2V8 ^®, SaniSure, of Moorpark, California, USA) and the contents of the vials is then pumped directly to the bioreactor through sterile welded C-flex tubing with a peristaltic pump. In preparation for planting pluripotent stem cells in a bioreactor, 0.15 L of E8 ™ medium was prepared with the addition of 0.5% BSA containing 10 μmol / L of Rho-kinase inhibitor, Y-27632, warmed to 37 ° C, mixed at 70 revolutions per minute, adjusted to 6.8-7.1 pH with CO ₂ , with a level of dissolved oxygen of 30% (content of CO ₂ , air, O ₂ and N _{2 is} regulated). Immediately after seeding into the bioreactor, samples were taken from it to count the cells, and the average volume was adjusted as necessary to obtain a final cell concentration of 0.225 × 10 ⁶ cells / ml.

Cells in a mechanically agitated bioreactor formed clusters in a mechanically agitated bioreactor. After seeding, the cell clusters were maintained in the reactor in an E8 ™ medium supplemented with 0.5% BSA for three days. The medium was replaced daily; 24 hours after seeding, 90% of the used medium was removed and 0.15 liters of fresh medium was added. After 48 after sowing, 90% of the used medium was removed and 0.15 L of fresh medium was added. 72 hours after seeding, differentiation of pluripotent cells was initiated by removing> 90% of the medium used and adding differentiation medium (Table 18).

After the start of the differentiation process, the cells were maintained for 12 or more days in a closed sterile suspension in a bioreactor with controlled temperature (37 ° C), pH 7.4 (for differentiation) with a set level of dissolved oxygen (10% DO at stages 1 and 30 % DO at other stages, the content of CO ₂ , O ₂ , N ₂ and air is regulated). Throughout the entire differentiation process, with each medium change, the impeller was stopped for 5–20 minutes before the medium was removed through a dip tube to stabilize the clusters. The medium in the bioreactor was removed or added from / to a closed vial or sachet by a peristaltic pump through a dip tube connected to tubes C-Flex ^® using Welder Terumo ™ tubing to maintain a closed system. The impeller and heater were restarted after adding sufficient medium to the vessel to immerse the wheel completely.

In order to control the process of the bioreactor, medium samples containing the cell clusters were collected daily to determine cell number and viability (NucleoCounter ^®), as shown in FIG. 45. In the process, total cell growth was observed, inoculating 0.225 × 10 ⁶ viable cells / ml grew to 0.65 × 10 ⁶ viable cells / ml at stage 4, day 3 (Fig. 45).

In addition to daily counts, bioreactor media samples were analyzed by NOVA BioProfile ^® FLEX (Nova Biomedical Corporation, Waltham, Mass., USA). It was noted that, relative to the set acidity of the reactor at the beginning of stage 0 (pH 6.8), the pH of the medium during stage 0 became more acidic (pH 6.8) (Fig.46). The initial acidity settings at stage 0 seemed to reduce the metabolic activity of the cells, with relatively low levels of lactic acid and high glucose levels, which were observed in the medium at stage 0. As soon as the cells began to differentiate in the second half of stage 3, the cells absorbed almost all of the glucose Fig.47) in the medium and began to generate a high level of lactic acid (Fig.48). In addition, during stages 1 and 2, an increase in cell density was observed (Fig.45).

In order to determine whether the stage-specific changes in pH and metabolism correspond to the stepwise changes in mRNA expression patterns measured by Qrt-PCR, the following was made. Four Applied Biosystems Low Density arrays (Life ™, Carlsbad, California, USA) labeled “pluripotency”, “definitive endoderm (DE)”, “intestinal tube” (GT) or “4 stage (S4)” were used. The results are presented as multiple differences with respect to undifferentiated UTCiPS cell samples, where undifferentiated cells act as controls to standardize the expression of all runs and arrays.

Using these arrays, gene expression was determined at each stage of differentiation. It was then that it became noticeable that the seed material of the thawed cells in the bioreactor showed undifferentiated patterns of gene expression in stage 0 days 1, 2, and 3 (24, 48 and 72 hours after seeding the bioreactor: Fig. 49 and 50). These results correlate well with flow cytometry, which showed high levels of expression of CD9, SSEA4, TRA-1-60, TRA- and 1-81, as well as the absence of CXCR4 / CD184 (Figure 51). Flow cytometry results and Qrt-PCR data showed robust and stable expression patterns of pluripotency genes (CD9, NANOG, POU5F1, SOX2, TDGF and ZFP42) and lack of expression of genes that are characteristically expressed during differentiation (CD99, CDH2, CDX2, CER1, CXCR4 , EOMES, FGF17, FGF4, Foxa2, GATA2, GATA4, GATA6, RCG Hand2, HNF4α KIT, MNX1, MIXL1, PRDM1, PTHR1R, SOX17, SOX7, T, TMPRSS2 and FV), characterizing the stable pluripotent state.

At the end of stage 0 (72 hours after seeding the reactor), the cells were transferred to differentiation medium (Table 18) containing MCX and GDF8. Twenty-four hours after this replacement of the medium, the authors noted significant changes in the structure of gene expression (Fig. 49 and 50, the fold increase in expression compared to undifferentiated control), for example,> 10-fold increase in the expression of FOXA2, HAND2, PRDM1, PTH1R and SOX17 ,> 100-fold increase in expression of CER1, FGF4, GATA4, GATA6, RCG and MNX1 and> 1000-fold increase in expression of EOMES, FGF17, MIXL1 and T. These increased expression levels indicated that the cells passed the mesodermal stage. It was also noted that CDX2 levels increased at stage 1 day 1 compared to undifferentiated cells (2700x increase in expression compared to control), however, this transient increase in expression and CDX2 expression fell by 97% during stage 1 day 3 to levels comparable to that observed prior to the induction of differentiation (Figs. 49 and 50, a fold increase in expression compared to undifferentiated control).

72 hours after contact with stage 1 differentiation medium, the cells expressed a profile according to the specification for definitive endoderm, and CXCR4 levels reached ~ 400x increase compared with control reference data, Foxa2 expression reached 136x increase compared to reference control data and SOX17 expression reached 470,000x increase compared with reference control data. In accordance with the characteristics of the definitive endoderm, it was also noted that the expression of the CER1, EOMES, FGF4, GSC, MIXL1 and T genes at the end of stage 1 (day 3) decreased from elevated levels observed at stage 1 and day 1 (Fig. 49 and 50 , the multiplicity of increase in expression compared to undifferentiated control).

These changes in gene expression observed with Qrt-PCR correlate with the results obtained using flow cytometry. There was an almost complete transition from the CD9-expressing / CXCR4-negative population of pluripotent cells when differentiation was initiated (Fig. 51) to a homogeneous population of cells expressing CXCR4 (98.3% of CXCR4-positive cells) at the end of stage 1 (Fig. 52).

After the formation of the definitive endoderm (stage 1), the medium was replaced with another containing FGF7 morphogen, which is used to cause the formation of a primitive anterior intestine. In accordance with the formation of the primitive gut, the expression levels of HNF4α and GATA6 at stage 2 days 1 and 3 were increased, while the genes with high expression level at stage 1 day 3 (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX17 and VWF) showed a decrease in expression by the end of stage 2 (Fig. 50 and 53, the multiplicity of the increase in expression compared to the undifferentiated control). The expression of the genes of the anterior gut (AFP, HHEX, Pdx1 and Prox1) was increased (Fig. 53, fold increase in expression compared to the undifferentiated control).

After the cells were cultured in medium during stage 2 for 72 hours, the culture was transferred to stage 3 medium (Table 18). After introducing cell expression markers into this medium, they showed results characteristic of pancreatic endoderm, which was determined by Qrt-PCR analysis of gene expression. Pdx1 expression increased 60-fold, from 12,000x relative to control at the end of stage 2, 3 days and to 739,000x relative to control at the end of stage 3, 3 days. These data show that cells differentiate into pancreatic (Fig.54). This observation was also confirmed by increased expression levels, in comparison with undifferentiated control, of certain genes usually expressed in the pancreas (ARX, GAST, GCG, INS, Isl1, NEUROD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, PAX6, Ptf1a and SST), as shown in FIG. 54 and 55. Interestingly, OCT4 / POU5F1 expression was not observed (37 samples of Cts tested using qRT-PCR), but high levels of expression of other markers of endodermal AFP, ALB, and CDX2 lines were observed. This indicates that the cell populations in the bioreactor were differentiated from a population of pluripotent cells, first into cells with respect to the plastic intestinal tube, and then into pancreatic cells (Fig. 54 and 55).

At the end of stage 4 of the differentiation process, the cells maintained a high level of Pdx1 expression (95.6% Pdx1-positive, by FACS, ~ 1000000 times induced compared with the control according to Qrt-PCR) and Foxa2 (99.5% Foxa2-positive by FACS) . Cells showed expression patterns corresponding to those of pancreatic progenitor cells (39.2% of Nkx6.1-positive by FACS) and the population of pancreatic endocrine cells (9.4% of PAX6-positive, 12.4% of chromagranin-positive, 15.2 % NKX2.2 positive; all by FACS). Patterns of marker expression characteristic for this stage indicate an effective stepwise differentiation from the population of pluripotent cells into progenitor cells of pancreatic cells. These results, observed in flow cytometry, were confirmed by Qrt-PCR. It was also noted that all genes commonly expressed in the pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST) showed increased expression level at the stage of 4 days 3. (Fig. 55). For reference, in FIG. 56 shows a representative micrograph (4x) of cell clusters at the end of each stage.

Table 18 Original day / date Stage 1 Stage 2 Stage 3 Stage 4 Base environment MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) MCDB131 Adapt.
(3.64 g / l NaCO ₃ ) Supplements 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1:50 000 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
1: 200 ITS-X
Glutamax 1: 100 Growth factors Only day 1 and 2:
GDF8
100 ng / ml FGF7
50 ng / ml FGF7
50 ng / ml not Small molecules Only day 1:
MCX
[2 μm] RA [2 μM]
SANT [0.25 μM]
TPPB [100 nM]
Only day 1
LDN 100 SANT [0.25 μM]
TPPB [100 nM] Day 3 3 3 3 NOTES :
All days relative to 0 h. Medium change
Day 1 and 2,
No replacement for day 3 Medium change
Day 1 and 3,
No replacement for day 2 Medium change
Day 1 and 2,
No replacement for day 3 In the case of S4 growth, replace the medium on day 1 and by the end of day 3

Table 18a BX Replication Seed material CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 one KC 83.3 0.1 99.9 94.5 85,8 2 Hw 95.5 0.2 100 91 84 3 ISM (pink) 95,8 0.1 100 76.1 36.5 four ISM (pink) 93.2 0 99.9 78.6 64.5 five ISM 1 97,8 0.2 99 74.8 66.4 6 ISM 2 98.6 0.2 100 92.2 86 7 ISM 1 98.1 0.1 99.9 88,8 80.3 eight ISM 1 99.1 0.1 99.9 93,8 83.3 9 ISM 2 97.2 0.1 99.9 88.3 81 ten ISM5 98 0.1 99.3 93.1 85.7 eleven ISM6 72.6 0.2 99.9 94.7 88.9 12 ISM6 85.9 0.7 99.4 71.9 54.1 CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 The average 93.6 0.1 99.8 87,8 76.6 Art. deviation 8.3 0.1 0.3 7,6 15.5

Table 18b Stage-day-time The density of viable cells (million cells / ml) CD9 CD184 SSEA4 TRA-1-60 TRA-1-81 S0D3-24H 0.626 95,8 0.1 99.8 87.9 74 The density of viable cells (million cells / ml) CD9 CD184 CD99 S1D3-24H 0.9 50.7 98.9 99 Viable cell density
(million cells / ml) NKX6.1 Khoromatogranin NKX2.2 PDX1 FOXA2 S4D1-24H 0.943 69.3 14.2 23.6 98.8 99.7 Viable cell density
(million cells / ml) NKX6.1 Khoromatogranin CDX2 SOX2 NKX2.2 PD PDX1 FOX FOXA2 NEU NEUROD S4D3-24H 1,002 66.2 35.6 0.3 15.8 38.1 9 99 9 99 45. 45,6

Materials:

• Cells of the human embryonic cell line H1 (hES), (cells WA01, WiCell, Madison WI)

• PBS (Cat. No. 14190, Invitrogen)

• Y-27632 (Axxora Part No. ALX-270-333, San Diego, CA)

• EDTA, (Lonza, catalog number 17-7-11E)

• NucleoCounter ^® - (ChemoMetec A / S, catalog number YC-T100, Allerod, Denmark)

• Processed 6-well plates with two non-woven cultures (Becton Dickinson, Catalog No. Falcon 351146, Franklin Lakes, NJ)

• Accutase ^® , (Sigma-Aldrich, Part No. A-6964, St. Louis, Missouri, USA)

• Bioreactor sensors for determining pH and dissolved oxygen (DO) (FermProbe ^® pH electrode 225 mm, model number F-635, OxyProbe ^® dissolved oxygen sensor 12 mm, model number D-145 from Broadley-James Corporation, Irwin state of California, USA)

• An immuno-protective macroencapsulation device (TheraCyte ™, Irvine, California, USA)

• ELISA of human C-peptide (MERCODIA; Cat. No. 10-1141-01)

• GlutaMAX ™, MCDB131 and Invitrogen ITS-X

• FAF-BSA (Proliant)

• Retinoic acid, glucose 45% (2.5M), SANT (Shh inhibitor) (Sigma)

• GDF8 (Peprotech)

• MCX

• FGF7 (R & D Systems)

• LDN-193189 (BMP receptor antagonist) (Stemgent)

• TPPB (PKC activator) (ChemPartner)

Example 15

Differentiation of embryonic stem cells from the WA01 cell line into definitive endoderm: the role of MCX / GDF8 as a regulator of the cell cycle in suspension culture

Clusters of pluripotent embryonic human stem cells from the H1 stem cell line (NIH code: WA01) were seeded at 0.5 × 10 ⁶ cells / ml in Erlenmeyer flasks with stirring on MCDB-131 medium containing 3.64 g / ml sodium bicarbonate and 5 , 5 mM glucose (Catalog No. A13051 DJ, Invitrogen, CA), which was supplemented with 2% 2% BSA without fatty acids (Catalog No. 68700, Proliant, I. A.), 1X GlutaMAX ™ (Catalog No. 35050- 079, Invitrogen, California), an additional 2.5 mM glucose (catalog number G8769, Sigma) and ITS-x at an initial concentration of 1: 50,000 (catalog number 51500056, Invitrogen, CA). The MCDB-131 medium, supplemented in this way, will be referred to as the base medium of stage 1 or the “Clean” medium for the purposes of this example. GSK3B inhibitor, 14-prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ .1 ~ 8.12 ~] heptacosis-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-nonen-16-one, US patent application No. 12 / 494,789; incorporated herein by reference in full, called MCX.

Clusters were treated on the first day of differentiation with one of six conditions: (1) pure, (2) 3 μmol MCX plus 100 ng / ml GDF-8 (Catalog No. 120-00, Peprotech), (3) 3 μmol only MCX, (4) 100 ng / ml GDF-8 only, (5) 20 ng / ml WNT-3A (catalog number 1324-WN-002, R & D Systems, Minnesota, USA) plus 100 ng / ml activin A (no. 338-AC, R & D Systems, Minnesota, USA) or (6) 20 ng / ml of WNT-3A only.

The medium in each of the conditions was replaced 24 and 48 hours after the start of differentiation. At this time, in cells according to conditions 1, 2, 3 and 4, the medium was replaced with fresh basic medium of stage 1 with the addition of 100 ng / ml GDF8, and in cells according to conditions 5 and 6 the medium was replaced with fresh basic medium of stage 1 by adding 100 ng / ml activin A.

One hour before the start of the differentiation and 5, 23, 29, 47 or 71 hours after the start of differentiation (referred to as "time 0") slurry samples were transferred to the treated two non-woven cultures six-well plates and incubated with EdU (Click-iT ^® EdU Kit, Life Technologies Corporation, Carlsbad, California, USA) for one hour. Incubated with EdU cells were then assessed using flow cytometry at 0, 6, 24, 30, 48, or 72 h after the start of differentiation to measure the percentage of cells at stages G0 / G1, S, or G2 / M of the cell cycle (Fig.81- 87).

According to this protocol, there were significant differences in the percentage of cells in the G0 / G1, S, or G2 / M stages of the cell cycle (Fig. 82-87), and it was noted that cells treated with MCX and MCX + GDF8 showed almost 40% the reduction of EdU inclusions compared with the other four processing conditions (Fig. 81). This decrease in EDU inclusions was accompanied by a 38% increase in the number of G0 / G1 cells in a sample treated with MCX + GDF8 and an increase of 54% in the number of G0 / G1 cells in a sample treated with MCX only. These changes in EDU inclusions and an increase in the transition to G0 / G1 6 hours after the start of differentiation was not observed in cells treated with GDF8, WNT3A, WNT-3A + activin A or pure medium. Rather, cells treated with GDF8, WNT-3A, WNT-3A + activin A or pure medium showed a minimal decrease in the percentage of cells with EDU inclusions (mean 48.1%, SD ± 1.2) and an average of 13% decrease in the number of cells in G0 / G1 six hours after the start of differentiation (standard deviation, ± 5%), as shown in Fig.81 and 82.

Similar differences were observed later in the distribution process between the G0 / G1 values for cells treated with MCX or MCX + GDF8 compared to other treatment conditions. 30 hours after time 0, it was found that cells treated with MCX or MCX + GDF8 showed 43-45% fewer cells in G0 / G1, compared with cells treated with WNT-3A + activin A, GDF8, WNT-3A, or clean environment. This gap between the percentage of G0 / G1 cells was unchanged 48 hours after the start of differentiation, 71.9-75.5% of cells treated with MCX or MCX + GDF8 were observed at the stages of the G0 / G1 cell cycle, with 48.5% for GDF8, 55.8% for WNT3A, 57.7% for Wnt-3A + activin A, and 49% for pure media. In addition to the observed differences in EDU inclusions and G0 / G1 profiles, cells treated with MCX or MCX + GDF8 resulted in 15–33% more cells in the S phase of the cell cycle after 30 and 48 hours after time 0, compared with cells treated with WNT3a + activin, GDF8, WNT-3A or pure medium (Fig.84 and 85).

Data (gene expression CD99, CD9, CDH1, CDH2, CDX2, CER1, CXCR4, FGF17, FGF4, Foxa2, GATA4, GATA6, GCG, KIT, MIXL1, MNX1, NANOG, Otx2, POU5F1, SOX17, SOX7 and T are shown on 57-80 and 88a-f) showed that in suspension culture, the addition of MCX with or without the substance of the TGF-β family, GDF8, on the first days of differentiation causes the generation of definitive endoderm, comparable to that of cell treatment with 20 ng / ml WNT- 3A +100 ng / ml of activin A on the first day based on the results of measurement of gene expression at the end of definitive endoderm formation. However, in accordance with the differences in the cell cycle observed during the formation of the definitive endoderm, intermediate differences in gene expression were observed. In samples treated with MCX or MCX + GDF8, T (Brachyury), GATA4 and CDX2 genes were induced at a level significantly higher than that of cells treated with Wnt-3A + activin A or three other tested conditions during the first 24 hours of differentiation ( Figures 88 B, C, and D). On the other hand, expression of pluripotency genes (NANOG and POU5F1 / OCT4) was significantly reduced after 24 hours in samples treated with MCX or MCX + GDF8, compared with the original cell population or other four test conditions (Fig.88E). The magnitude of induction of gene expression, such as FGF4, Foxa2 and SOX17, was significantly lower in MCX or MCX + GDF8 samples, compared to the other four conditions tested, within 24 hours after differentiation began, but after 48 hours, FGF4, Foxa2 and SOX17 expression all samples had comparable levels. (Fig. 88c and e).

Example 16

Generation of ectodermal and mesodermal tissue using a differentiation process in a scalable suspension.

This example demonstrates a process during which both growth and differentiation of pluripotent stem cells (PSC) occurs and thereby achieves a scalable manufacturing process for generating ectodermal or mesodermal tissue.

Two cell lines were grown in suspension to provide seed for these studies: sub-clone H1 (WA01) of the hES WB0106 cell line and induced pluripotent stem cell line (iPSC) generated from umbilical cord tissue (UTC) cells. As described in previous examples, cells grown in suspension were frozen at high density in a freezer at a controlled rate, then thawed for seeding in a closed 3-liter glass bioreactor (DASGIP; Jülich, Germany) or a disposable 3-liter bioreactor (Mobius ^® , EMD Millipore Corporation, Billerica, Mass., USA) at a final cell concentration of 0.225 × 10 ⁶ cells / ml. Cells that were seeded into a mechanically agitated bioreactor formed clusters of cells in a continuous mixing tank and kept in pluripotency medium (E8 ™ with addition of 0.5% BSA) in the reactor for three days. 72 hours after seeding, differentiation of pluripotent cells was initiated by transferring cell clusters to plastic Erlenmeyer flasks (125 ml PETG flasks, Catalog No. 4112, Thermo Scientific Rochester New York) into the appropriate differentiation medium (Table 19), to form mesoderm cardiac tissue (1) or ectoderm / nervous tissue (2).

After the start of the differentiation process, the cells were maintained for ten (10) days at 100 revolutions per minute in a humidified 5% CO2 incubator on a moving platform (MAXQ 416hp, Thermo Scientific, Rochester New York). On day 1, day 3, day 5 and day 7 after the start of differentiation, the medium in the flask was replaced with fresh medium, as shown in Table 19. Before the differentiation began, the samples were subjected to qRT-PCR analysis to obtain reference values, and then the procedure was repeated on 3, 5, 7 and 10 days after the start of differentiation.

To check whether it is possible to determine the presence of ectodermal or mesodermal specific changes in mRNA expression patterns using QRT-PCR, three Applied Biosystems Low Density arrays (Life ™, Carlsbad, Calif.), Labeled "Pluripotency", "The definitive endoderm (DE ) ”And“ Stage 6 (S6) ”, and the results were compared with data from samples of the corresponding undifferentiated pluripotent stem cells as a control for standardization of expression.

Using these arrays, gene expression patterns of pluripotent cells cultured in ectodermal (Fig.89) or mesodermal (Fig.90) differentiation media are determined. It was noted that cells differentiated in stirred flasks, treated according to any of the conditions, showed a decrease in the expression of pluripotency genes, such as NANOG, POU5F1 / OCT4, TDGF1 and ZFP42, as the culture grew from 3 days to 10 days, which was measured on array of pluripotency. CXCR4 expression increased in HES or iPS cells differentiated into the ectoderm or mesoderm. These results correlate with Qrt-PCR data showing high expression of the genes characteristic of differentiation. Cells treated with ectodermal differentiation medium showed an increased level of expression of ARX, NeuroD, Nkx6.1, PAX6 (> 100 times), and ZIC1 (> 1000 times) according to Qrt-PCR from 3 to 10 days after the start of differentiation (Fig. 91 ). These data were confirmed by the FACS array, which showed that on the third (3) day after the start of differentiation into ectoderm cells, IPSC cells and hES maintained a high expression level of SOX2 (pluripotency gene and neural stem cells), but lost the expression of POU5F1 / OCT4 (genes pluripotency), but the expression of PAX6 (a gene of nervous and endocrine differentiation) was revealed (Fig.92).

Similar differentiation kinetics were observed in cells treated with medium for mesodermal differentiation. While expression of pluripotent genes decreased during the 10-day differentiation (Fig. 90), on day 3, early induction of genes characteristic of the early transitional stage of differentiation of the mesoderm (CER1, EOMES, CKIT and VWF) was observed, the level of expression of these genes on the tenth day dropped to almost the original (Fig. 93). It was also noted that the expression of genes characteristic of the mesoderm at 3, 5, 7 and 10 days after the beginning of differentiation showed early high expression of the genes (CDH2, CDX2, GATA6, HNF4α, MNX1, PRDM1 and SOX17 in Fig. 93). The same pattern of gene expression induction was observed in both IPS and HES cell samples, indicating that the differentiation process was directional and not spontaneous in nature.

These changes in gene expression observed with Qrt-PCR correlate with the results obtained using phase-contrast microscopy and immune staining of clusters subjected to cryosection. On the 10th day in the mesodermal differentiated suspension culture, approximately 1 out of 10 clusters began to “shrink” spontaneously, which suggested that the cells differentiated into myocardial tissue (Fig.94, left panel, 10th day, white stripes). A colored cross section of some clusters showed a continuous layered pattern with β-tubulin stained, which indicates the formation of muscle tissue (Fig. 94, right).

The morphological picture observed for clusters differentiated into ectodermal cells (Fig. 95, left) is very different compared with clusters differentiated into mesodermal cells (Fig.94). Clusters in the whole volume of ectodermal differentiation were larger and denser than cells differentiated into mesodermal cells, and in cells differentiated into ectodermal cells, β-tubulin expression was less pronounced. Cells showing β-tubulin expression showed a more dendritic pattern when stained (Fig. 95, right panel, white arrows), characteristic of neurons.

These results, in combination with Qrt-PCR and FACS data, show that cells deposited in a bank and grown in suspension can be differentiated in suspension culture into mesodermal or ectodermal cells in a directed and reproducible manner.

Table 19 Start
Day / Date Neuronal differentiation
Day 0-4 Neuronal differentiation
Day 5-10 Cardiac differentiation
Day 0-6 Cardiac differentiation
Day 7-10 Base environment MCDB131
(2.5 g / l NaCO ₃ final) MCDB131 Adapt.
(2.5 g / l NaCO ₃ final) MCDB131 Adapt.
(2.5 g / l NaCO ₃ final) MCDB131 Adapt.
(2.5 g / l NaCO ₃ ) Supplements 2% FAF-BSA
2.5 mM glucose
Glutamax 1: 100
1: 100 ITS-X 2% FAF-BSA
2.5 mM glucose
Glutamax 1: 100
1: 100 ITS-X or
1X B-27 2% FAF-BSA
2.5 mM glucose
Glutamax 1: 100 2% FAF-BSA
2.5 mM glucose
Glutamax 1: 100
1X B-27 Small molecules LDN 100
ALKVi [7.5 μm] missing Only the first 24 hours
MCX [2 μM]
Only day 3 and 4:
IWP-4 [8 μm] Day 3 3 3 3 NOTES :
Every day are counted from the start time Replacing the environment:
Day 0, 1 and 3 Replacing the environment:
Day 5 and 7 Replacing the environment:
Day 0, 1, 3 and 5 Medium change
Day 7

Table 20 Materials: cells derived from human umbilical cord tissue (as disclosed in US Pat. No. 7,510,873) Induced pluripotent stem cells Parthenots Cells of the human embryonic cell line H1 (hES), (cells WA01, WiCell, Madison WI) PBS (Cat. No. 14190, Invitrogen) Y-27632 (Axxora Catalog Number ALX-270-333, San Diego, CA) EDTA, (Lonza, catalog number 17-7-11E) NucleoCounter®- (ChemoMetec A / S, catalog number YC-T100, Allerod, Denmark) 6-well plates treated with two non-woven cultures (Becton Dickinson, Catalog No. Falcon 351146, Franklin Lakes, NJ) Accutase ^® , (Sigma, catalog No. A-6964, St. Louis, Missouri, USA) Sensors for determining the level of bioreactor pH and amounts of dissolved oxygen (DO) (FermProbe® pH electrode is 225 mm, the model № F-635, dissolved oxygen sensor OxyProbe ^® 12 mm, model number D-145 from Broadley-James Corporation, Irvine state California, USA) Immuno-Macro-Encapsulation Device (TheraCyte ™, Irwin, California, USA) ELISA of human C-peptide (MERCODIA Part No. 10-1141-01) GlutaMAX ™, MCDB131 and ITS-X (Life Technologies Corporation, Grand Island, NY, USA) FAF-BSA (Proliant) Retinoic acid, glucose 45% (2.5M), SANT (Shh inhibitor) (Sigma) GDF8 (Peprotech) MCX IWP-4 (WNT3 inhibitor) Stemgent Wednesday MCDB131 Wednesday MCDB131 (adapted (MCDB131 adapt.)) - modified to increase the level of NaCO ₃ to 3.64 g / l.

While the invention has been described and illustrated with reference to various specific materials, procedures, and examples, it should be understood that the invention is not limited to specific combinations of material and procedures selected for this purpose. Numerous changes regarding such details of the invention will be apparent to those skilled in the art. It is understood that all descriptions and examples are presented only as an illustration and are within the essence and scope of the present invention, which is reflected in the following claims. All references, patents and patent applications cited in this application are fully incorporated into this document by reference.

Claims (51)

1. In vitro method of obtaining three-dimensional cell clusters from pluripotent stem cells, including: a. treatment of pluripotent stem cells cultured in a flat adhesive culture together with a chelating agent or enzyme, with the release of cell aggregates from the flat adhesive culture; b. suspending cell aggregates from a flat adhesive culture into the culture medium in the presence of an Rho kinase inhibitor without dissociating cell aggregates to single cells; c. transfer of cell aggregate suspension to dynamic suspension culture; and d. an increase in the volume of cell aggregate suspension in a dynamic suspension culture with obtaining three-dimensional cell clusters, while three-dimensional cell clusters retain pluripotency, moreover, pluripotent stem cells are induced pluripotent stem cells, cells derived from human umbilical cord tissue, parthenotes, cells derived from amniotic fluid, or human embryonic stem cells of the H1, H7, H9, SA002 or BG01v human stem cells. 2. A method according to claim 1, wherein the pluripotent stem cells are treated with an enzyme selected from a neutral protease or Accutase®. 3. The method according to p. 2, in which the enzyme is a neutral protease. 4. The method according to claim 3, wherein the pluripotent stem cells are H1 hES. 5. The methods of claim 1, wherein the cells in the cell clusters express CD9, SSEA4, TRA-1-60 and TRA-1-81 and do not express CXCR4. 6. A method according to claim 1, wherein the pluripotent stem cells are treated with a chelating agent. 7. A method according to claim 6, in which the chelating agent is ethylenediaminetetraacetic acid (EDTA). 8. The method according to claim 1, wherein the dynamically stirred suspension culture contains microcarriers. 9. The method of differentiation of cell clusters of pluripotent stem cells in a dynamically mixed suspension culture system, including: but. treatment of pluripotent stem cells cultured in a flat adhesive culture together with a chelating agent or enzyme, with the release of cell aggregates from the flat adhesive culture; b. suspending cell aggregates from a flat adhesive culture in the presence of an Rho kinase inhibitor without dissociating cell aggregates to single cells; with. transfer of cell aggregate suspension to a dynamically stirred suspension culture; d. increasing the volume of cell aggregate suspension in a dynamically stirred suspension culture to produce pluripotent cell clusters in which cell clusters express CD9, SSEA4, TRA-1-60 and TRA-1-81 and do not express CXCR4; and e. differentiation of pluripotent cell clusters in a dynamic suspension culture system to produce a population of pancreatic progenitor cells, a population of neuron progenitor cells, or a population of cardiomyocyte progenitor cells, moreover, pluripotent stem cells are induced pluripotent stem cells, cells derived from human umbilical cord tissue, parthenotes, cells derived from amniotic fluid, or human embryonic stem cells of the H1, H7, H9, SA002 or BG01v human stem cells. 10. The method of claim 9, wherein a population of pancreatic progenitor cells is produced which express the transcription factors of β-cells. 11. The method according to p. 10, in which the transcription factors of β-cells are PDX1 and / or NKX6.1. 12. The method according to p. 9, which includes the differentiation of pluripotent cell clusters in a dynamically mixed suspension culture system to obtain a population of progenitor cells of the pancreas. 13. The method according to claim 9, which includes the differentiation of pluripotent cell clusters in a dynamically mixed suspension culture system to obtain a population of neuron precursor cells. 14. The method according to p. 9, which includes the differentiation of pluripotent cell clusters in a dynamically mixed suspension culture system to obtain a population of precursor cells of cardiomyocytes. 15. The method according to claim 9, wherein the pluripotent stem cells are treated with a chelating agent. 16. The method according to claim 15, in which the chelating agent is EDTA. 17. The method according to claim 9, wherein the pluripotent stem cells are treated with an enzyme. 18. The method according to p. 17, in which the enzyme is a neutral protease or Accutase. 19. The method according to p. 9, in which the stage of differentiation includes cultivation when the oxygen level is from about hypoxia to about 30% of ambient air, lipids in the range from 0.1% to about 2%, or a combination of these conditions. 20. A method according to claim 12, which includes the differentiation of three-dimensional pluripotent stem cell clusters into final endoderm cells in a culture medium supplemented with either (i) cyclic aniline pyridinotriazine and GDF8, or (ii) WNT3A and activin A. 21. The method according to claim 20, wherein the cyclic anilinpyridinotriazine is 14-prop-2-en-1-yl-1-3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1 ~ 2, 6 ~ .1 ~ 8,12 ~] - [heptacosis-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-nona-16-one. 22. The method according to p. 9, in which the dynamically stirred suspension culture system contains microcarriers. 23. In vitro method for producing pluripotent stem cell clusters, including: but. treatment of pluripotent stem cells cultured in a flat adhesive culture, together with an enzymatic or chelating agent at room temperature for a period of time sufficient to release the pluripotent stem cells in the form of cell aggregates; b. removing the specified enzymatic or chelating agent; with. adding the culture medium with the addition of the Rho-kinase inhibitor to the cell aggregates and suspending the cell aggregates into the culture without dissociating the cell aggregates into single cells; d. transfer of cell aggregate suspension to a dynamic suspension culture system; and e. an increase in the volume of the suspension of cell aggregates in a dynamic suspension culture with the production of cell clusters, while cells in cell clusters are pluripotent, moreover, pluripotent stem cells are induced pluripotent stem cells, cells derived from human umbilical cord tissue, parthenotes, cells derived from amniotic fluid, or human embryonic stem cells of the H1, H7, H9, SA002 or BG01v human stem cells. 24. A method according to claim 23, in which the method includes the use of chelating agents. 25. The method according to claim 24, in which the chelating agent is EDTA. 26. The method according to p. 25, in which the method includes the use of enzymatic agents. 27. The method according to p. 23, in which the enzymatic agent is a neutral protease or Accutase. 28. The method of claim 23, further comprising differentiating pluripotent stem cell clusters in a dynamic suspension culture system to produce a population of pancreatic progenitor cells, a population of neuronal progenitor cells, or a population of cardiomyocyte progenitor cells. 29. The method according to claim 1, wherein the Rho kinase inhibitor is Y-27632. 30. The method according to claim 1, wherein the culture medium contains an inhibitor of Rho-kinase at a concentration of from 1 to about 100 microns. 31. The method of claim 9, wherein the Rho kinase inhibitor is Y-27632. 32. The method of claim 9, wherein the cell aggregates from the flat adhesive culture are suspended in the presence of from 1 to about 100 μM Rho kinase inhibitor. 33. The method of claim 23, wherein the Rho kinase inhibitor is Y-27632. 34. The method according to p. 23, in which the culture medium contains an inhibitor of Rho-kinase at a concentration of from 1 to about 100 microns.

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