Limits...
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

Characterization of iPSCs derived from PBMC and cord blood. A subset of iPSC clones was characterized. The experiments demonstrated in the figure provide representative examples of the results observed for iPSC clone 1, 2, 3 derived from peripheral blood donor PL#10, and iPSC clone 1, 2, 3 derived from cord donor CB1. a, PBMC derived-iPSCs show typical human ESC morphology express pluripotent markers OCT4, SOX2, SSEA4, NANOG detected by immunochemistry. b, Representative cytogenetic analysis on G-banded metaphase cells from iPSC clone 1 exhibiting a normal karyotype. iPSCs can form EB, spontaneously differentiate towards ectoderm (Tuj1), mesoderm (SMA), and endoderm (SMA), and endoderm (ALB). c, Approximately 3000 cord blood cells were efficiency reprogrammed. Picked colony shows typical human ESC morphology, express plurinent cell surface marker TRA-1-60, patient disease ELK3 gene mutations conforming the sample identity. d, Cord blood derived iPSCs can form EB efficiently, spontaneous differentiation results show their differentiation potential towards ectoderm (NESTIN, Tuj1), mesoderm (SMA), and endoderm (AFP). e, Pluripotent gene expression determined by quantifying RNA transcripts counts using nCounter Analysis System, error bars represent standard derivation of the mean (n = 2). f, Hierarchical Cluster of EBs. g, Detection of sendal viral vector by immunohistochemistry
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4493720&req=5

Fig5: Characterization of iPSCs derived from PBMC and cord blood. A subset of iPSC clones was characterized. The experiments demonstrated in the figure provide representative examples of the results observed for iPSC clone 1, 2, 3 derived from peripheral blood donor PL#10, and iPSC clone 1, 2, 3 derived from cord donor CB1. a, PBMC derived-iPSCs show typical human ESC morphology express pluripotent markers OCT4, SOX2, SSEA4, NANOG detected by immunochemistry. b, Representative cytogenetic analysis on G-banded metaphase cells from iPSC clone 1 exhibiting a normal karyotype. iPSCs can form EB, spontaneously differentiate towards ectoderm (Tuj1), mesoderm (SMA), and endoderm (SMA), and endoderm (ALB). c, Approximately 3000 cord blood cells were efficiency reprogrammed. Picked colony shows typical human ESC morphology, express plurinent cell surface marker TRA-1-60, patient disease ELK3 gene mutations conforming the sample identity. d, Cord blood derived iPSCs can form EB efficiently, spontaneous differentiation results show their differentiation potential towards ectoderm (NESTIN, Tuj1), mesoderm (SMA), and endoderm (AFP). e, Pluripotent gene expression determined by quantifying RNA transcripts counts using nCounter Analysis System, error bars represent standard derivation of the mean (n = 2). f, Hierarchical Cluster of EBs. g, Detection of sendal viral vector by immunohistochemistry

Mentions: In addition, the generation of iPSC free of integrated reprogramming factor genes is essential to reduce differentiation biases and artificial phenotype [24]. Immunochemistry staining with anti-Sendai antibody was negative for Sendai virus vector in all test colonies that were obtained at passage 5 (Fig. 5g), confirmed that Sendai virus vector were diluted out during the proliferation, and no transgene were carried in the iPSCs.Fig. 5


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)

Characterization of iPSCs derived from PBMC and cord blood. A subset of iPSC clones was characterized. The experiments demonstrated in the figure provide representative examples of the results observed for iPSC clone 1, 2, 3 derived from peripheral blood donor PL#10, and iPSC clone 1, 2, 3 derived from cord donor CB1. a, PBMC derived-iPSCs show typical human ESC morphology express pluripotent markers OCT4, SOX2, SSEA4, NANOG detected by immunochemistry. b, Representative cytogenetic analysis on G-banded metaphase cells from iPSC clone 1 exhibiting a normal karyotype. iPSCs can form EB, spontaneously differentiate towards ectoderm (Tuj1), mesoderm (SMA), and endoderm (SMA), and endoderm (ALB). c, Approximately 3000 cord blood cells were efficiency reprogrammed. Picked colony shows typical human ESC morphology, express plurinent cell surface marker TRA-1-60, patient disease ELK3 gene mutations conforming the sample identity. d, Cord blood derived iPSCs can form EB efficiently, spontaneous differentiation results show their differentiation potential towards ectoderm (NESTIN, Tuj1), mesoderm (SMA), and endoderm (AFP). e, Pluripotent gene expression determined by quantifying RNA transcripts counts using nCounter Analysis System, error bars represent standard derivation of the mean (n = 2). f, Hierarchical Cluster of EBs. g, Detection of sendal viral vector by immunohistochemistry
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4493720&req=5

Fig5: Characterization of iPSCs derived from PBMC and cord blood. A subset of iPSC clones was characterized. The experiments demonstrated in the figure provide representative examples of the results observed for iPSC clone 1, 2, 3 derived from peripheral blood donor PL#10, and iPSC clone 1, 2, 3 derived from cord donor CB1. a, PBMC derived-iPSCs show typical human ESC morphology express pluripotent markers OCT4, SOX2, SSEA4, NANOG detected by immunochemistry. b, Representative cytogenetic analysis on G-banded metaphase cells from iPSC clone 1 exhibiting a normal karyotype. iPSCs can form EB, spontaneously differentiate towards ectoderm (Tuj1), mesoderm (SMA), and endoderm (SMA), and endoderm (ALB). c, Approximately 3000 cord blood cells were efficiency reprogrammed. Picked colony shows typical human ESC morphology, express plurinent cell surface marker TRA-1-60, patient disease ELK3 gene mutations conforming the sample identity. d, Cord blood derived iPSCs can form EB efficiently, spontaneous differentiation results show their differentiation potential towards ectoderm (NESTIN, Tuj1), mesoderm (SMA), and endoderm (AFP). e, Pluripotent gene expression determined by quantifying RNA transcripts counts using nCounter Analysis System, error bars represent standard derivation of the mean (n = 2). f, Hierarchical Cluster of EBs. g, Detection of sendal viral vector by immunohistochemistry
Mentions: In addition, the generation of iPSC free of integrated reprogramming factor genes is essential to reduce differentiation biases and artificial phenotype [24]. Immunochemistry staining with anti-Sendai antibody was negative for Sendai virus vector in all test colonies that were obtained at passage 5 (Fig. 5g), confirmed that Sendai virus vector were diluted out during the proliferation, and no transgene were carried in the iPSCs.Fig. 5

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