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Shin Kawamata

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George Sandusky

John Dillberger

Anna Migliaccio

The University of Chicago Medical Center

Toshime Igarashi

Universidad Cooperativa de Colombia

Mark Hoenerhoff

University of Michigan

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Papers by Shin Kawamata

Data on long-term survival of the NOD/Shi-scid IL-2Rγnull (NOG) mouse in two facilities

The Journal of Toxicological Sciences

The objective of this study was to investigate an appropriate observation period for an evaluatio... more The objective of this study was to investigate an appropriate observation period for an evaluation of tumorigenicity in NOD/Shi-scid IL-2 Rγ null (NOG) mice. At SNBL, 19 male and 19 female NOG mice were observed the general condition from 7 weeks old up to 68 weeks old and at FBRI, 7 male and 16 female NOG mice were observed the general condition throughout the lifespan from 7 weeks old. The survival rate started to decline rapidly around 54 to 56 weeks of age in both facilities without a facility difference. Based on these survival data, it seems reasonable to terminate a tumorigenicity study at 52 weeks of age.

Divergent Point of Developmental Cell Fate of Common Precursor for Cardiomyocyte and Hemangioblast Is Accompanied by the Presence or Absence of Expression of PDGFRalpha and Flk-1

Blood

Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular an... more Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular and molecular mechanisms regulating germ layer fate determination and tissue formation. Both of hemangioblasts and cardiomyocytes are thought to be derived from lateral plate mesoderm of early embryo, but little is known about the phenotypic specification in these two lines of differentiation at the divergent point through development. Utilizing the in vitro differentiation system of murine ES cells in the presence or absence of stromal cell layer, and gene expression data in the microarray analysis, we determined a set of cell-surface molecules as specific markers, which enabled efficient enrichment of hemangioblasts or cardiac myocytes. Furthermore, we represent the evidences that the sorted fraction with hemangioblast-specific markers markedly produced both of blood island and endotherial colonies in stromal-free culture system. Materials and Methods Murine embryonic stem cell line, EB5...

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017

STEM CELLS

Quality control guidelines for clinical-grade human induced pluripotent stem cell lines

Regenerative medicine, 2018

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for... more Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

Report of the international conference on manufacturing and testing of pluripotent stem cells

Biologicals

Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

Scientific reports, Jan 10, 2018

Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to dif... more Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to differentiate. Here we show the differentiation potential and growth rate of ESC correlates positively with the expression level of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). When ESCs are maintained in feeder-free conditions and single cell seeding, ESC KhES-1 having 4520 copies or more of CHD7 in 5 ng total RNA show differentiation potential, but this is lost when the CHD7 copy number is reduced in KhES-1 to less than 696 by alternative culture conditions. Introduction of siCHD7 reduced differentiation potential and growth rate of KhES-1. Interestingly, KhES-1 underwent spontaneous differentiation when mCHD7 was introduced and we could not obtain CHD7-overexpressing ESC in culture. These data suggest that CHD7 drives differentiation, and there is a lower limit for CHD7 to initiate differentiation and an upper limit for CHD7 if maintained in undifferentiated stat...

Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells

Stem cells translational medicine, 2017

This article summarizes the recent activity of the International Stem Cell Banking Initiative (IS... more This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Transla...

Identification of a Gene Encoding Slow Skeletal Muscle Troponin T as a Novel Marker for Immortalization of Retinal Pigment Epithelial Cells

Scientific reports, Jan 15, 2017

Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeut... more Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in AR...

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration

The New England journal of medicine, Mar 16, 2017

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells di... more We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] nu...

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products

Biologicals : journal of the International Association of Biological Standardization, Jan 22, 2016

The development of human cell therapy and gene therapy products has progressed internationally. E... more The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is neede...

Method for evaluating multipotent stem cells by image analysis

Positive imaging of cardiomyopathy with 99m Tc(V)-DMSA

Ann Nucl Med, 1992

A case of histologically proven dilated cardiomyopathy and a case of clinically diagnosed cardiom... more A case of histologically proven dilated cardiomyopathy and a case of clinically diagnosed cardiomyopathy (cardiac amyloidosis was strongly suspected but was not confirmed) were examined with 99mTc(V)-dimercaptosuccinic acid (DMSA). 99mTc(V)-DMSA accumulation in the damaged myocardium was clearly demonstrated. These results suggested the possibility that 99mTc(V)-DMSA could be used as a positive agent for cardiomyopathy.

Coexpression of platelet-derived growth factor receptor alpha and fetal liver kinase 1 enhances cardiogenic potential in embryonic stem cell differentiation in vitro

Journal of Bioscience and Bioengineering, May 31, 2007

Intracellular Signaling of GP34, the OX40 Ligand: Induction of C-Jun and C-Fos Mrna Expression Through GP34 Upon Binding of Its Receptor, OX40

The Journal of Immunology, Sep 15, 1999

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF fam... more We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.

An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions

American journal of stem cells, 2015

Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condi... more Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condition can facilitate metabolome and proteome analysis of culturing cells and medium, and reduce regulatory concerns for clinical application of cells. And in addition, if hPSC are passaged and cryopreserved in single cells it also facilitates quality control of cells at single cell level. Here we report a robust single cell freezing and thawing method of hPSCs cultured in chemically-defined medium TeSR(TM)-E8(TM) and on cost-effective recombinant human Vitronectin-N (rhVTN-N)-coated dish. Cells are dissociated into single cells with recombinant TrypLE(TM) Select and 0.5 mM EDTA/PBS (3:1 solution) in the presence of Rock inhibitor and cryopreserved with chemically defined CryoStem(TM). Approximately 60% of cells were viable after dissociation. Aggrewell(TM) 400 was used to form cell clumps of 500 cells after thaw in the presence of Rock inhibitor and cells were cultured for two days with T...

Method of treatment for ischemic heart disease

Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

Regenerative Therapy, 2015

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

Scientific reports, Jan 8, 2015

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human... more Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay sa...

System for Injecting Liquid Drug Containing Biological Material

Incidence of tumor formation after transplanting HeLa cells in various immunodeficient mice

Data on long-term survival of the NOD/Shi-scid IL-2Rγnull (NOG) mouse in two facilities

The Journal of Toxicological Sciences

The objective of this study was to investigate an appropriate observation period for an evaluatio... more The objective of this study was to investigate an appropriate observation period for an evaluation of tumorigenicity in NOD/Shi-scid IL-2 Rγ null (NOG) mice. At SNBL, 19 male and 19 female NOG mice were observed the general condition from 7 weeks old up to 68 weeks old and at FBRI, 7 male and 16 female NOG mice were observed the general condition throughout the lifespan from 7 weeks old. The survival rate started to decline rapidly around 54 to 56 weeks of age in both facilities without a facility difference. Based on these survival data, it seems reasonable to terminate a tumorigenicity study at 52 weeks of age.

Divergent Point of Developmental Cell Fate of Common Precursor for Cardiomyocyte and Hemangioblast Is Accompanied by the Presence or Absence of Expression of PDGFRalpha and Flk-1

Blood

Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular an... more Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular and molecular mechanisms regulating germ layer fate determination and tissue formation. Both of hemangioblasts and cardiomyocytes are thought to be derived from lateral plate mesoderm of early embryo, but little is known about the phenotypic specification in these two lines of differentiation at the divergent point through development. Utilizing the in vitro differentiation system of murine ES cells in the presence or absence of stromal cell layer, and gene expression data in the microarray analysis, we determined a set of cell-surface molecules as specific markers, which enabled efficient enrichment of hemangioblasts or cardiac myocytes. Furthermore, we represent the evidences that the sorted fraction with hemangioblast-specific markers markedly produced both of blood island and endotherial colonies in stromal-free culture system. Materials and Methods Murine embryonic stem cell line, EB5...

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017

STEM CELLS

Quality control guidelines for clinical-grade human induced pluripotent stem cell lines

Regenerative medicine, 2018

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for... more Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

Report of the international conference on manufacturing and testing of pluripotent stem cells

Biologicals

Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

Scientific reports, Jan 10, 2018

Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to dif... more Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to differentiate. Here we show the differentiation potential and growth rate of ESC correlates positively with the expression level of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). When ESCs are maintained in feeder-free conditions and single cell seeding, ESC KhES-1 having 4520 copies or more of CHD7 in 5 ng total RNA show differentiation potential, but this is lost when the CHD7 copy number is reduced in KhES-1 to less than 696 by alternative culture conditions. Introduction of siCHD7 reduced differentiation potential and growth rate of KhES-1. Interestingly, KhES-1 underwent spontaneous differentiation when mCHD7 was introduced and we could not obtain CHD7-overexpressing ESC in culture. These data suggest that CHD7 drives differentiation, and there is a lower limit for CHD7 to initiate differentiation and an upper limit for CHD7 if maintained in undifferentiated stat...

Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells

Stem cells translational medicine, 2017

This article summarizes the recent activity of the International Stem Cell Banking Initiative (IS... more This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Transla...

Identification of a Gene Encoding Slow Skeletal Muscle Troponin T as a Novel Marker for Immortalization of Retinal Pigment Epithelial Cells

Scientific reports, Jan 15, 2017

Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeut... more Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in AR...

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration

The New England journal of medicine, Mar 16, 2017

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells di... more We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] nu...

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products

Biologicals : journal of the International Association of Biological Standardization, Jan 22, 2016

The development of human cell therapy and gene therapy products has progressed internationally. E... more The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is neede...

Method for evaluating multipotent stem cells by image analysis

Positive imaging of cardiomyopathy with 99m Tc(V)-DMSA

Ann Nucl Med, 1992

A case of histologically proven dilated cardiomyopathy and a case of clinically diagnosed cardiom... more A case of histologically proven dilated cardiomyopathy and a case of clinically diagnosed cardiomyopathy (cardiac amyloidosis was strongly suspected but was not confirmed) were examined with 99mTc(V)-dimercaptosuccinic acid (DMSA). 99mTc(V)-DMSA accumulation in the damaged myocardium was clearly demonstrated. These results suggested the possibility that 99mTc(V)-DMSA could be used as a positive agent for cardiomyopathy.

Coexpression of platelet-derived growth factor receptor alpha and fetal liver kinase 1 enhances cardiogenic potential in embryonic stem cell differentiation in vitro

Journal of Bioscience and Bioengineering, May 31, 2007

Intracellular Signaling of GP34, the OX40 Ligand: Induction of C-Jun and C-Fos Mrna Expression Through GP34 Upon Binding of Its Receptor, OX40

The Journal of Immunology, Sep 15, 1999

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF fam... more We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.

An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions

American journal of stem cells, 2015

Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condi... more Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condition can facilitate metabolome and proteome analysis of culturing cells and medium, and reduce regulatory concerns for clinical application of cells. And in addition, if hPSC are passaged and cryopreserved in single cells it also facilitates quality control of cells at single cell level. Here we report a robust single cell freezing and thawing method of hPSCs cultured in chemically-defined medium TeSR(TM)-E8(TM) and on cost-effective recombinant human Vitronectin-N (rhVTN-N)-coated dish. Cells are dissociated into single cells with recombinant TrypLE(TM) Select and 0.5 mM EDTA/PBS (3:1 solution) in the presence of Rock inhibitor and cryopreserved with chemically defined CryoStem(TM). Approximately 60% of cells were viable after dissociation. Aggrewell(TM) 400 was used to form cell clumps of 500 cells after thaw in the presence of Rock inhibitor and cells were cultured for two days with T...

Method of treatment for ischemic heart disease

Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

Regenerative Therapy, 2015

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

Scientific reports, Jan 8, 2015

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human... more Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay sa...

System for Injecting Liquid Drug Containing Biological Material

Incidence of tumor formation after transplanting HeLa cells in various immunodeficient mice