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Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.

Zhou H, Martinez H, Sun B, Li A, Zimmer M, Katsanis N, Davis EE, Kurtzberg J, Lipnick S, Noggle S, Rao M, Chang S - Stem Cell Rev (2015)

Bottom Line: The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports.Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way.In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

View Article: PubMed Central - PubMed

Affiliation: The New York Stem Cell Foundation Research Institute, New York, NY, 10032, USA, mzhou@nyscf.org.

ABSTRACT
Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

No MeSH data available.


Related in: MedlinePlus

Directed differentiation of blood cell-derived iPSCs. Representative examples of the results observed for iPSC clone 3 derived from peripheral blood donor PL#10, and iPSC clone 1 derived from cord blood donor CB1. a, NSC differentiated from iPSCs. Cells stained with neural stem cell specific markers SOX1, SOX2 and NESTIN. b, Cardiomyocyte differentiated from iPSCs, cell stained with markers α-Actinin (ACT). NKX2.5, Troponin T (TnT), and DAPI. c, Hepatocyte-like cells differentiated from iPSC. Cells stained with α-fetoprotein (AFP), albumin (ALB), and DAPI. d, Periodic acid-Schiff (PAS) staining. Purple color in cells indicates glycogen accumulation. e, Albumin secretion by CB_iPSC derived hepato-cyte-like cells was assayed by ELISA. The differentiation medium was changed to fresh medium 24 h before the assay. The concentration of the ALB secreted from 30 k iPSC_derived hepatocyte-like cells in 200 ul of media was measured on differentiation Day 16. Error bars represent the standard deviation of the mean (N = 2)
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Fig6: Directed differentiation of blood cell-derived iPSCs. Representative examples of the results observed for iPSC clone 3 derived from peripheral blood donor PL#10, and iPSC clone 1 derived from cord blood donor CB1. a, NSC differentiated from iPSCs. Cells stained with neural stem cell specific markers SOX1, SOX2 and NESTIN. b, Cardiomyocyte differentiated from iPSCs, cell stained with markers α-Actinin (ACT). NKX2.5, Troponin T (TnT), and DAPI. c, Hepatocyte-like cells differentiated from iPSC. Cells stained with α-fetoprotein (AFP), albumin (ALB), and DAPI. d, Periodic acid-Schiff (PAS) staining. Purple color in cells indicates glycogen accumulation. e, Albumin secretion by CB_iPSC derived hepato-cyte-like cells was assayed by ELISA. The differentiation medium was changed to fresh medium 24 h before the assay. The concentration of the ALB secreted from 30 k iPSC_derived hepatocyte-like cells in 200 ul of media was measured on differentiation Day 16. Error bars represent the standard deviation of the mean (N = 2)

Mentions: We applied a multistage differentiation protocol developed previously to promote the conversion of human iPSCs into NSCs [18, 19] via inhibition of TGFβ and BMP signaling in a defined cell monolayer system. The treatments resulted in the emergence of typical NSCs that express marker proteins such as SOX1, NESTIN, and SOX2 at a similar efficiency across the iPSC lines (95 ± 5.6 %) (Fig. 6a).Fig. 6


Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.

Zhou H, Martinez H, Sun B, Li A, Zimmer M, Katsanis N, Davis EE, Kurtzberg J, Lipnick S, Noggle S, Rao M, Chang S - Stem Cell Rev (2015)

Directed differentiation of blood cell-derived iPSCs. Representative examples of the results observed for iPSC clone 3 derived from peripheral blood donor PL#10, and iPSC clone 1 derived from cord blood donor CB1. a, NSC differentiated from iPSCs. Cells stained with neural stem cell specific markers SOX1, SOX2 and NESTIN. b, Cardiomyocyte differentiated from iPSCs, cell stained with markers α-Actinin (ACT). NKX2.5, Troponin T (TnT), and DAPI. c, Hepatocyte-like cells differentiated from iPSC. Cells stained with α-fetoprotein (AFP), albumin (ALB), and DAPI. d, Periodic acid-Schiff (PAS) staining. Purple color in cells indicates glycogen accumulation. e, Albumin secretion by CB_iPSC derived hepato-cyte-like cells was assayed by ELISA. The differentiation medium was changed to fresh medium 24 h before the assay. The concentration of the ALB secreted from 30 k iPSC_derived hepatocyte-like cells in 200 ul of media was measured on differentiation Day 16. Error bars represent the standard deviation of the mean (N = 2)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Directed differentiation of blood cell-derived iPSCs. Representative examples of the results observed for iPSC clone 3 derived from peripheral blood donor PL#10, and iPSC clone 1 derived from cord blood donor CB1. a, NSC differentiated from iPSCs. Cells stained with neural stem cell specific markers SOX1, SOX2 and NESTIN. b, Cardiomyocyte differentiated from iPSCs, cell stained with markers α-Actinin (ACT). NKX2.5, Troponin T (TnT), and DAPI. c, Hepatocyte-like cells differentiated from iPSC. Cells stained with α-fetoprotein (AFP), albumin (ALB), and DAPI. d, Periodic acid-Schiff (PAS) staining. Purple color in cells indicates glycogen accumulation. e, Albumin secretion by CB_iPSC derived hepato-cyte-like cells was assayed by ELISA. The differentiation medium was changed to fresh medium 24 h before the assay. The concentration of the ALB secreted from 30 k iPSC_derived hepatocyte-like cells in 200 ul of media was measured on differentiation Day 16. Error bars represent the standard deviation of the mean (N = 2)
Mentions: We applied a multistage differentiation protocol developed previously to promote the conversion of human iPSCs into NSCs [18, 19] via inhibition of TGFβ and BMP signaling in a defined cell monolayer system. The treatments resulted in the emergence of typical NSCs that express marker proteins such as SOX1, NESTIN, and SOX2 at a similar efficiency across the iPSC lines (95 ± 5.6 %) (Fig. 6a).Fig. 6

Bottom Line: The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports.Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way.In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

View Article: PubMed Central - PubMed

Affiliation: The New York Stem Cell Foundation Research Institute, New York, NY, 10032, USA, mzhou@nyscf.org.

ABSTRACT
Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

No MeSH data available.


Related in: MedlinePlus