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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

Seeding density and attachment effect on peripheral blood reprogramming. a, upper panel: representative live whole-well TRA-1-60 images from wells of 24-well plates containing different seeding number. After 4 days cell expansion, the cell in suspension were collected for reprogramming induction, and named as Suspended Cells; the cell attached to the plate bottom were dissociated with 0.5 mM EDTA and transfected with reprogramming factors and termed as Adherent Cells. Images were recorded by Celigo software 10 days post reprogramming factors transduction. Lower panel: reprogramming efficiency for each peripheral blood samples. Efficiency was calculated based on total TRA-1-60 colony count from both suspended cells and adherent cells for each seeding density. b, upper panel: representative live whole-well TRA-1-60 images from wells of 96-well plates containing different seeding number. Three kinds of hESC medium were tested in the reprogramming assay (Freedome-1, E8, and E6+bFGF), images were recorded by Celigo software 10 days post reprogramming factor transduction. Lower panel: reprogramming efficiency for each condition was calculated based on the total TRA-1-60 colony count
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Fig2: Seeding density and attachment effect on peripheral blood reprogramming. a, upper panel: representative live whole-well TRA-1-60 images from wells of 24-well plates containing different seeding number. After 4 days cell expansion, the cell in suspension were collected for reprogramming induction, and named as Suspended Cells; the cell attached to the plate bottom were dissociated with 0.5 mM EDTA and transfected with reprogramming factors and termed as Adherent Cells. Images were recorded by Celigo software 10 days post reprogramming factors transduction. Lower panel: reprogramming efficiency for each peripheral blood samples. Efficiency was calculated based on total TRA-1-60 colony count from both suspended cells and adherent cells for each seeding density. b, upper panel: representative live whole-well TRA-1-60 images from wells of 96-well plates containing different seeding number. Three kinds of hESC medium were tested in the reprogramming assay (Freedome-1, E8, and E6+bFGF), images were recorded by Celigo software 10 days post reprogramming factor transduction. Lower panel: reprogramming efficiency for each condition was calculated based on the total TRA-1-60 colony count

Mentions: In our initial experiment, PBMCs were seeded on standard 24-well plates. The cells were cultured in the presence of IL3, IL6, SCF and FLT3 for 4 days to expand the hematopoietic progenitors, and transfected with Sendai virus encoding Oct4, Sox2, Klf4, and cMyc. After 2 days of recovery, the transduced blood cells were transferred onto Geltrex (Life Technologies) coated plates and cultured without cytokines for another 4 days. At Day 7, the cells were incubated with customized human ESC medium Freedom-1 (Life Technologies). The first iPSC colonies appeared approximately 1 week after transfection (Day 7, Fig. 1b). To determine the appropriate cell number needed for reprogramming, we set up a cell seeding density experiment in standard 24-well plates (25 to 200 k per well of 24-well plates). We found although most of the cells stayed in suspension, some cells spontaneously attached to the culture surface during the 4-day cell expansion (for instance, for PL#7, we observed more cells attached to the culture surface from 50 k seeding well than from 25 or 100 k seeding wells). Both the attached and suspended cells can be reprogrammed as demonstrated by the appearance of TRA-1-60+ colonies, though the reprogramming efficiency varies from sample to sample (0.15–0.32 %, Fig. 2a). We observed that initial seeding at 25–50 k per well of 24-well plates result in a uniform distribution of TRA-1-60 positive colonies, while there are too many colonies to distinguish if more than 100 k cells were seeded (Fig. 2a).Fig. 2


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)

Seeding density and attachment effect on peripheral blood reprogramming. a, upper panel: representative live whole-well TRA-1-60 images from wells of 24-well plates containing different seeding number. After 4 days cell expansion, the cell in suspension were collected for reprogramming induction, and named as Suspended Cells; the cell attached to the plate bottom were dissociated with 0.5 mM EDTA and transfected with reprogramming factors and termed as Adherent Cells. Images were recorded by Celigo software 10 days post reprogramming factors transduction. Lower panel: reprogramming efficiency for each peripheral blood samples. Efficiency was calculated based on total TRA-1-60 colony count from both suspended cells and adherent cells for each seeding density. b, upper panel: representative live whole-well TRA-1-60 images from wells of 96-well plates containing different seeding number. Three kinds of hESC medium were tested in the reprogramming assay (Freedome-1, E8, and E6+bFGF), images were recorded by Celigo software 10 days post reprogramming factor transduction. Lower panel: reprogramming efficiency for each condition was calculated based on the total TRA-1-60 colony count
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Related In: Results  -  Collection

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Fig2: Seeding density and attachment effect on peripheral blood reprogramming. a, upper panel: representative live whole-well TRA-1-60 images from wells of 24-well plates containing different seeding number. After 4 days cell expansion, the cell in suspension were collected for reprogramming induction, and named as Suspended Cells; the cell attached to the plate bottom were dissociated with 0.5 mM EDTA and transfected with reprogramming factors and termed as Adherent Cells. Images were recorded by Celigo software 10 days post reprogramming factors transduction. Lower panel: reprogramming efficiency for each peripheral blood samples. Efficiency was calculated based on total TRA-1-60 colony count from both suspended cells and adherent cells for each seeding density. b, upper panel: representative live whole-well TRA-1-60 images from wells of 96-well plates containing different seeding number. Three kinds of hESC medium were tested in the reprogramming assay (Freedome-1, E8, and E6+bFGF), images were recorded by Celigo software 10 days post reprogramming factor transduction. Lower panel: reprogramming efficiency for each condition was calculated based on the total TRA-1-60 colony count
Mentions: In our initial experiment, PBMCs were seeded on standard 24-well plates. The cells were cultured in the presence of IL3, IL6, SCF and FLT3 for 4 days to expand the hematopoietic progenitors, and transfected with Sendai virus encoding Oct4, Sox2, Klf4, and cMyc. After 2 days of recovery, the transduced blood cells were transferred onto Geltrex (Life Technologies) coated plates and cultured without cytokines for another 4 days. At Day 7, the cells were incubated with customized human ESC medium Freedom-1 (Life Technologies). The first iPSC colonies appeared approximately 1 week after transfection (Day 7, Fig. 1b). To determine the appropriate cell number needed for reprogramming, we set up a cell seeding density experiment in standard 24-well plates (25 to 200 k per well of 24-well plates). We found although most of the cells stayed in suspension, some cells spontaneously attached to the culture surface during the 4-day cell expansion (for instance, for PL#7, we observed more cells attached to the culture surface from 50 k seeding well than from 25 or 100 k seeding wells). Both the attached and suspended cells can be reprogrammed as demonstrated by the appearance of TRA-1-60+ colonies, though the reprogramming efficiency varies from sample to sample (0.15–0.32 %, Fig. 2a). We observed that initial seeding at 25–50 k per well of 24-well plates result in a uniform distribution of TRA-1-60 positive colonies, while there are too many colonies to distinguish if more than 100 k cells were seeded (Fig. 2a).Fig. 2

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