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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

TCR and IGH rearrangement analysis in PBMC and CB iPSCs. a, PCR analysis of TCRB rearrangements. b, PCR analysis of IGH rearrangements. c, PCR analysis of TCRG rearrangements. d, PCR analysis of TCRD rearrangements. e, Specimen control. IVS-0000: polyclonal control DNA; IVS-0004, IVS-0021, IVS-0030: clonal control DNA; H2O: no template control; Buffy coat: genomic DNA from buffy coat PL#10
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Fig4: TCR and IGH rearrangement analysis in PBMC and CB iPSCs. a, PCR analysis of TCRB rearrangements. b, PCR analysis of IGH rearrangements. c, PCR analysis of TCRG rearrangements. d, PCR analysis of TCRD rearrangements. e, Specimen control. IVS-0000: polyclonal control DNA; IVS-0004, IVS-0021, IVS-0030: clonal control DNA; H2O: no template control; Buffy coat: genomic DNA from buffy coat PL#10

Mentions: Since iPSCs carrying pre-existing gene rearrangement may have uncertain functional consequences [12, 23], we used PCR to detect clonal IGH and TCR gene rearrangements in iPSCs to determine if they were derived from B- or T- lymphocytes. As shown in Fig. 4, whereas the clonal control and parental whole blood cells showed bands reflecting the presence of gene rearrangements, all tested iPSC lines did not show bands in the valid size range for TCRB_Vβ + Jβ, Vβ + Jβ2, Dβ + Jβ (Fig. 4a), TCRG (Fig. 4c), TCRD (Fig. 4d), and IGH gene rearrangements (Fig. 4b), indicating they were derived from non-B, non-T cells. This could be explained by the selective cell growth of CD13+ and CD71+ cells with the cell expansion cocktail (Fig. 3a).Fig. 4


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)

TCR and IGH rearrangement analysis in PBMC and CB iPSCs. a, PCR analysis of TCRB rearrangements. b, PCR analysis of IGH rearrangements. c, PCR analysis of TCRG rearrangements. d, PCR analysis of TCRD rearrangements. e, Specimen control. IVS-0000: polyclonal control DNA; IVS-0004, IVS-0021, IVS-0030: clonal control DNA; H2O: no template control; Buffy coat: genomic DNA from buffy coat PL#10
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493720&req=5

Fig4: TCR and IGH rearrangement analysis in PBMC and CB iPSCs. a, PCR analysis of TCRB rearrangements. b, PCR analysis of IGH rearrangements. c, PCR analysis of TCRG rearrangements. d, PCR analysis of TCRD rearrangements. e, Specimen control. IVS-0000: polyclonal control DNA; IVS-0004, IVS-0021, IVS-0030: clonal control DNA; H2O: no template control; Buffy coat: genomic DNA from buffy coat PL#10
Mentions: Since iPSCs carrying pre-existing gene rearrangement may have uncertain functional consequences [12, 23], we used PCR to detect clonal IGH and TCR gene rearrangements in iPSCs to determine if they were derived from B- or T- lymphocytes. As shown in Fig. 4, whereas the clonal control and parental whole blood cells showed bands reflecting the presence of gene rearrangements, all tested iPSC lines did not show bands in the valid size range for TCRB_Vβ + Jβ, Vβ + Jβ2, Dβ + Jβ (Fig. 4a), TCRG (Fig. 4c), TCRD (Fig. 4d), and IGH gene rearrangements (Fig. 4b), indicating they were derived from non-B, non-T cells. This could be explained by the selective cell growth of CD13+ and CD71+ cells with the cell expansion cocktail (Fig. 3a).Fig. 4

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