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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

Derivation of human iPSCs from human peripheral blood samples. a Scheme for reprogramming human peripheral blood. b Live whole-well (24-well plates) and zoomed representative images of peripheral blood at different stages during reprogramming process. Images were taken by Celigo software. D-3: Three days before transduction; D7: Seven days after transduction; D 10: Ten days after transduction
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Fig1: Derivation of human iPSCs from human peripheral blood samples. a Scheme for reprogramming human peripheral blood. b Live whole-well (24-well plates) and zoomed representative images of peripheral blood at different stages during reprogramming process. Images were taken by Celigo software. D-3: Three days before transduction; D7: Seven days after transduction; D 10: Ten days after transduction

Mentions: Blood cell expansion medium contained StemPro-34 SFM (Life Technologies) supplemented with 100 ng/ml stem cell factor (SCF, R&D Systems), 100 ng/ml FLT3 (eBiosciences), 20 ng/ml interleukin-3 (IL3, Cell Signaling), and 20 ng/ml interleukin-6 (IL6, Cell Signaling). Medium was changed every day for 4 days (Day -4 to Day -1, Fig. 1a) by centrifugation to remove the medium and replacing with fresh medium. After 4 days cell expansion (Day 0), cells were transduced by Oct4, Sox2, Klf4, and c-Myc Sendai viral vectors (CytoTune-iPSC 2.0 Sendai Reprogramming Kit, Life Technologies) at a multiplicity of infection (MOI) of 5. The transduction was performed in StemPro-34 SFM supplemented with cytokines containing 4 μg/mL of Polybrene by centrifugation at 2000 RPM for 30 min. The day after transfection (Day 1), Sendai Viruses were removed by centrifuging the cell suspension. The cells were resuspended with fresh StemPro-34 SFM supplemented with cytokines for 2 days. The next day (Day 3), the cells were then collected by centrifugation, resuspended with StemPro-34 SFM without cytokines, and seeded onto Geltrex-coated plates at the targeted densities. The medium was refreshed every other day. From Day 6–7, the medium was changed to customized human ESC medium Freedom-1 (Life Technologies) with daily medium changes. Once the ESC like TRA-1-60+ iPSC emerged, the colonies were manually picked and replated onto Geltrex-coated plates for expansion. For extremely small number of cord blood cell reprogramming (e.g., 3000 cells), cells were reprogrammed using the same method as above except using a higher MOI of 15.Fig. 1


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)

Derivation of human iPSCs from human peripheral blood samples. a Scheme for reprogramming human peripheral blood. b Live whole-well (24-well plates) and zoomed representative images of peripheral blood at different stages during reprogramming process. Images were taken by Celigo software. D-3: Three days before transduction; D7: Seven days after transduction; D 10: Ten days after transduction
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493720&req=5

Fig1: Derivation of human iPSCs from human peripheral blood samples. a Scheme for reprogramming human peripheral blood. b Live whole-well (24-well plates) and zoomed representative images of peripheral blood at different stages during reprogramming process. Images were taken by Celigo software. D-3: Three days before transduction; D7: Seven days after transduction; D 10: Ten days after transduction
Mentions: Blood cell expansion medium contained StemPro-34 SFM (Life Technologies) supplemented with 100 ng/ml stem cell factor (SCF, R&D Systems), 100 ng/ml FLT3 (eBiosciences), 20 ng/ml interleukin-3 (IL3, Cell Signaling), and 20 ng/ml interleukin-6 (IL6, Cell Signaling). Medium was changed every day for 4 days (Day -4 to Day -1, Fig. 1a) by centrifugation to remove the medium and replacing with fresh medium. After 4 days cell expansion (Day 0), cells were transduced by Oct4, Sox2, Klf4, and c-Myc Sendai viral vectors (CytoTune-iPSC 2.0 Sendai Reprogramming Kit, Life Technologies) at a multiplicity of infection (MOI) of 5. The transduction was performed in StemPro-34 SFM supplemented with cytokines containing 4 μg/mL of Polybrene by centrifugation at 2000 RPM for 30 min. The day after transfection (Day 1), Sendai Viruses were removed by centrifuging the cell suspension. The cells were resuspended with fresh StemPro-34 SFM supplemented with cytokines for 2 days. The next day (Day 3), the cells were then collected by centrifugation, resuspended with StemPro-34 SFM without cytokines, and seeded onto Geltrex-coated plates at the targeted densities. The medium was refreshed every other day. From Day 6–7, the medium was changed to customized human ESC medium Freedom-1 (Life Technologies) with daily medium changes. Once the ESC like TRA-1-60+ iPSC emerged, the colonies were manually picked and replated onto Geltrex-coated plates for expansion. For extremely small number of cord blood cell reprogramming (e.g., 3000 cells), cells were reprogrammed using the same method as above except using a higher MOI of 15.Fig. 1

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