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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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Detection of pluripotency-associated factors in LV-iPSC-derived grafts. (A): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for cMYC expression by immunostaining with specific antibodies against cMYC (red) and insulin (green). Nuclei were counter-stained by DAPI (blue). (B): The levels of cMYC transcripts in the grafts were determined by real-time reverse-transcription polymerase chain reaction. RNA samples from T1D LV2- and T1D SV#A-derived grafts (n = 3 each) were used for the assay. (C): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for OCT4 expression by immunostaining with specific antibodies against OCT4 (red) and insulin (green). Nuclei were counterstained by DAPI (blue). (D): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts, as well as normal mouse pancreas, were assessed for pancreatic lipase expression by immunostaining with specific antibodies against lypase (red) and insulin (green). Nuclei were counterstained by DAPI (blue). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed iPSC; TGF-iPSC, transgene-free iPSC; T1D, type 1 diabetes mellitus.
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Figure 3: Detection of pluripotency-associated factors in LV-iPSC-derived grafts. (A): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for cMYC expression by immunostaining with specific antibodies against cMYC (red) and insulin (green). Nuclei were counter-stained by DAPI (blue). (B): The levels of cMYC transcripts in the grafts were determined by real-time reverse-transcription polymerase chain reaction. RNA samples from T1D LV2- and T1D SV#A-derived grafts (n = 3 each) were used for the assay. (C): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for OCT4 expression by immunostaining with specific antibodies against OCT4 (red) and insulin (green). Nuclei were counterstained by DAPI (blue). (D): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts, as well as normal mouse pancreas, were assessed for pancreatic lipase expression by immunostaining with specific antibodies against lypase (red) and insulin (green). Nuclei were counterstained by DAPI (blue). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed iPSC; TGF-iPSC, transgene-free iPSC; T1D, type 1 diabetes mellitus.

Mentions: To assess the underlying mechanism of notable tumorigenicity of LV-iPSC-derived pancreatic endoderm cells, we first analyzed the representative iPSC-derived grafts for expression of cMYC, OCT4, and pancreatic acinar cell marker lypase. cMYC-positive cells were frequently found in LV-iPSC-derived tumors, whereas no notable cMYC signal was found in TGF-iPSC-derived grafts (Fig. 3A). We also found a trend of increased cMYC expression in LV-iPSC-derived grafts (Fig. 3B). Conversely, OCT4-positive cells were rare. Only one graft from LV-iPSCs showed notable OCT4 expression in ductal cells (Fig. 3C). Although we found good lipase expression in normal mouse pancreas, none of the iPSC-derived grafts showed notable lipase expression (Fig. 3D).


Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration
Detection of pluripotency-associated factors in LV-iPSC-derived grafts. (A): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for cMYC expression by immunostaining with specific antibodies against cMYC (red) and insulin (green). Nuclei were counter-stained by DAPI (blue). (B): The levels of cMYC transcripts in the grafts were determined by real-time reverse-transcription polymerase chain reaction. RNA samples from T1D LV2- and T1D SV#A-derived grafts (n = 3 each) were used for the assay. (C): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for OCT4 expression by immunostaining with specific antibodies against OCT4 (red) and insulin (green). Nuclei were counterstained by DAPI (blue). (D): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts, as well as normal mouse pancreas, were assessed for pancreatic lipase expression by immunostaining with specific antibodies against lypase (red) and insulin (green). Nuclei were counterstained by DAPI (blue). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed iPSC; TGF-iPSC, transgene-free iPSC; T1D, type 1 diabetes mellitus.
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Related In: Results  -  Collection

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Figure 3: Detection of pluripotency-associated factors in LV-iPSC-derived grafts. (A): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for cMYC expression by immunostaining with specific antibodies against cMYC (red) and insulin (green). Nuclei were counter-stained by DAPI (blue). (B): The levels of cMYC transcripts in the grafts were determined by real-time reverse-transcription polymerase chain reaction. RNA samples from T1D LV2- and T1D SV#A-derived grafts (n = 3 each) were used for the assay. (C): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts in the kidney capsules were assessed for OCT4 expression by immunostaining with specific antibodies against OCT4 (red) and insulin (green). Nuclei were counterstained by DAPI (blue). (D): Sections of recovered LV-iPSC- and TGF-iPSC-derived grafts, as well as normal mouse pancreas, were assessed for pancreatic lipase expression by immunostaining with specific antibodies against lypase (red) and insulin (green). Nuclei were counterstained by DAPI (blue). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LV-iPSC, lentivirus-reprogrammed iPSC; TGF-iPSC, transgene-free iPSC; T1D, type 1 diabetes mellitus.
Mentions: To assess the underlying mechanism of notable tumorigenicity of LV-iPSC-derived pancreatic endoderm cells, we first analyzed the representative iPSC-derived grafts for expression of cMYC, OCT4, and pancreatic acinar cell marker lypase. cMYC-positive cells were frequently found in LV-iPSC-derived tumors, whereas no notable cMYC signal was found in TGF-iPSC-derived grafts (Fig. 3A). We also found a trend of increased cMYC expression in LV-iPSC-derived grafts (Fig. 3B). Conversely, OCT4-positive cells were rare. Only one graft from LV-iPSCs showed notable OCT4 expression in ductal cells (Fig. 3C). Although we found good lipase expression in normal mouse pancreas, none of the iPSC-derived grafts showed notable lipase expression (Fig. 3D).

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