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Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration

View Article: PubMed Central - PubMed

ABSTRACT

Transplantation of progenitors from induced pluripotent stem cells reprogrammed by lentiviral vectors led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Combined transgene-free reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo.

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Combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny and regeneration of human islets from T1D patients. (A): Summary of the incidence of tumor formation upon transplantation of pancreatic endoderm cells from TGF- and LV-iPSCs, made by Sendai viral and lentiviral reprogramming vectors, respectively. Mice were observed for 3 months for the TGF-iPSC progeny, differentiated with the enzymatic dissociation protocol. Mice having received pancreatic endoderm cells from LV-iPSCs or TGF-iPSCs, underwent differentiation with the enzymatic or nonenzymatic dissociation protocols, respectively. Recipients, consequently, were sacrificed at 3-5 weeks post transplantation due to enlarged iPSC-derived tumors. (B): Sections of recovered TGF-iPSC-derived grafts in the kidney capsules were assessed for formation of human islet-like structures by immunostaining with specific antibodies against insulin, glucagon, PDX1 and human C-peptide. Nuclei were counterstained by DAPI (blue). (C): Representative immunostaining images of TGF-iPSC-derived islets with heterogeneity in the predominance of β- and α-cell contents from a single recipient mouse are shown. (D): Regeneration of human islets from a patient with T1D. SCID beige mice received pancreatic progenitor cells derived from TGF-iPSCs originating from a skin biopsy. Recovered grafts were assessed for islet regeneration by insulin and glucagon antibodies. Nuclei were counter stained by DAPI (white). (E): Circulating levels of human C-peptide was measured at 2 months after transplantation of iPSC-derived pancreatic progenitor cells. Plasma samples were harvested at fasting (pre) and 60 min after glucose challenge (post) conditions to measure human C-peptide levels by ELISA, an assay without cross-reactivity toward mouse C-peptide. No statistical significant glucose-responsive insulin secretion was observed. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed induced pluripotent stem cell; PBMC, peripheral blood mononuclear cell; TGF-iPSC, transgene-free induced pluripotent stem cell; T1D, type 1 diabetes mellitus.
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Figure 2: Combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny and regeneration of human islets from T1D patients. (A): Summary of the incidence of tumor formation upon transplantation of pancreatic endoderm cells from TGF- and LV-iPSCs, made by Sendai viral and lentiviral reprogramming vectors, respectively. Mice were observed for 3 months for the TGF-iPSC progeny, differentiated with the enzymatic dissociation protocol. Mice having received pancreatic endoderm cells from LV-iPSCs or TGF-iPSCs, underwent differentiation with the enzymatic or nonenzymatic dissociation protocols, respectively. Recipients, consequently, were sacrificed at 3-5 weeks post transplantation due to enlarged iPSC-derived tumors. (B): Sections of recovered TGF-iPSC-derived grafts in the kidney capsules were assessed for formation of human islet-like structures by immunostaining with specific antibodies against insulin, glucagon, PDX1 and human C-peptide. Nuclei were counterstained by DAPI (blue). (C): Representative immunostaining images of TGF-iPSC-derived islets with heterogeneity in the predominance of β- and α-cell contents from a single recipient mouse are shown. (D): Regeneration of human islets from a patient with T1D. SCID beige mice received pancreatic progenitor cells derived from TGF-iPSCs originating from a skin biopsy. Recovered grafts were assessed for islet regeneration by insulin and glucagon antibodies. Nuclei were counter stained by DAPI (white). (E): Circulating levels of human C-peptide was measured at 2 months after transplantation of iPSC-derived pancreatic progenitor cells. Plasma samples were harvested at fasting (pre) and 60 min after glucose challenge (post) conditions to measure human C-peptide levels by ELISA, an assay without cross-reactivity toward mouse C-peptide. No statistical significant glucose-responsive insulin secretion was observed. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed induced pluripotent stem cell; PBMC, peripheral blood mononuclear cell; TGF-iPSC, transgene-free induced pluripotent stem cell; T1D, type 1 diabetes mellitus.

Mentions: When TGF-iPSC clones were differentiated into pancreatic progenitor cells, by using the same differentiation protocol with nonenzymatic dissociation steps (Fig. 1A) and transplanted beneath the kidney capsule, TGF-iPSC grafts developed teratoma-like tumors (Fig. 2A). Because human pluripotent stem cells are sensitive to enzymatic single cell dissociation [32, 33], we used trypsinization-mediated cell dissociation to eliminate contamination by pluripotent cells of the iPSC-derived progeny population. Transplantation of the TGF-iPSC progeny, differentiated and enzymatically dissociated, resulted in no tumor formation at 3 and 8 months after transplantation (0 of 29 and 0 of 3 recipient mice with tumor) (Fig. 2A). In sharp contrast, enzymatic dissociation did not prevent tumor formation of LV-iPSC-derived pancreatic endoderm cells (Fig. 2A; 3 of 3 recipient mice transplanted with T1D-specific LV-iPSC progeny).


Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration
Combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny and regeneration of human islets from T1D patients. (A): Summary of the incidence of tumor formation upon transplantation of pancreatic endoderm cells from TGF- and LV-iPSCs, made by Sendai viral and lentiviral reprogramming vectors, respectively. Mice were observed for 3 months for the TGF-iPSC progeny, differentiated with the enzymatic dissociation protocol. Mice having received pancreatic endoderm cells from LV-iPSCs or TGF-iPSCs, underwent differentiation with the enzymatic or nonenzymatic dissociation protocols, respectively. Recipients, consequently, were sacrificed at 3-5 weeks post transplantation due to enlarged iPSC-derived tumors. (B): Sections of recovered TGF-iPSC-derived grafts in the kidney capsules were assessed for formation of human islet-like structures by immunostaining with specific antibodies against insulin, glucagon, PDX1 and human C-peptide. Nuclei were counterstained by DAPI (blue). (C): Representative immunostaining images of TGF-iPSC-derived islets with heterogeneity in the predominance of β- and α-cell contents from a single recipient mouse are shown. (D): Regeneration of human islets from a patient with T1D. SCID beige mice received pancreatic progenitor cells derived from TGF-iPSCs originating from a skin biopsy. Recovered grafts were assessed for islet regeneration by insulin and glucagon antibodies. Nuclei were counter stained by DAPI (white). (E): Circulating levels of human C-peptide was measured at 2 months after transplantation of iPSC-derived pancreatic progenitor cells. Plasma samples were harvested at fasting (pre) and 60 min after glucose challenge (post) conditions to measure human C-peptide levels by ELISA, an assay without cross-reactivity toward mouse C-peptide. No statistical significant glucose-responsive insulin secretion was observed. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed induced pluripotent stem cell; PBMC, peripheral blood mononuclear cell; TGF-iPSC, transgene-free induced pluripotent stem cell; T1D, type 1 diabetes mellitus.
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Figure 2: Combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny and regeneration of human islets from T1D patients. (A): Summary of the incidence of tumor formation upon transplantation of pancreatic endoderm cells from TGF- and LV-iPSCs, made by Sendai viral and lentiviral reprogramming vectors, respectively. Mice were observed for 3 months for the TGF-iPSC progeny, differentiated with the enzymatic dissociation protocol. Mice having received pancreatic endoderm cells from LV-iPSCs or TGF-iPSCs, underwent differentiation with the enzymatic or nonenzymatic dissociation protocols, respectively. Recipients, consequently, were sacrificed at 3-5 weeks post transplantation due to enlarged iPSC-derived tumors. (B): Sections of recovered TGF-iPSC-derived grafts in the kidney capsules were assessed for formation of human islet-like structures by immunostaining with specific antibodies against insulin, glucagon, PDX1 and human C-peptide. Nuclei were counterstained by DAPI (blue). (C): Representative immunostaining images of TGF-iPSC-derived islets with heterogeneity in the predominance of β- and α-cell contents from a single recipient mouse are shown. (D): Regeneration of human islets from a patient with T1D. SCID beige mice received pancreatic progenitor cells derived from TGF-iPSCs originating from a skin biopsy. Recovered grafts were assessed for islet regeneration by insulin and glucagon antibodies. Nuclei were counter stained by DAPI (white). (E): Circulating levels of human C-peptide was measured at 2 months after transplantation of iPSC-derived pancreatic progenitor cells. Plasma samples were harvested at fasting (pre) and 60 min after glucose challenge (post) conditions to measure human C-peptide levels by ELISA, an assay without cross-reactivity toward mouse C-peptide. No statistical significant glucose-responsive insulin secretion was observed. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed induced pluripotent stem cell; PBMC, peripheral blood mononuclear cell; TGF-iPSC, transgene-free induced pluripotent stem cell; T1D, type 1 diabetes mellitus.
Mentions: When TGF-iPSC clones were differentiated into pancreatic progenitor cells, by using the same differentiation protocol with nonenzymatic dissociation steps (Fig. 1A) and transplanted beneath the kidney capsule, TGF-iPSC grafts developed teratoma-like tumors (Fig. 2A). Because human pluripotent stem cells are sensitive to enzymatic single cell dissociation [32, 33], we used trypsinization-mediated cell dissociation to eliminate contamination by pluripotent cells of the iPSC-derived progeny population. Transplantation of the TGF-iPSC progeny, differentiated and enzymatically dissociated, resulted in no tumor formation at 3 and 8 months after transplantation (0 of 29 and 0 of 3 recipient mice with tumor) (Fig. 2A). In sharp contrast, enzymatic dissociation did not prevent tumor formation of LV-iPSC-derived pancreatic endoderm cells (Fig. 2A; 3 of 3 recipient mice transplanted with T1D-specific LV-iPSC progeny).

View Article: PubMed Central - PubMed

ABSTRACT

Transplantation of progenitors from induced pluripotent stem cells reprogrammed by lentiviral vectors led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Combined transgene-free reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo.

No MeSH data available.


Related in: MedlinePlus