Fig 1.
iPSC pool purification scheme using Magnetic Activated Cell Sorting (MACS) of TRA-1-60 or SSEA4 expressing cells.
Colonies from a duplicate reprogramming plate were also manually picked and expanded in parallel to establish clonally derived iPSC lines.
Fig 2.
Generation of iPSC pool by MACS from hSTEMCCA reprogrammed cells.
Cell populations from before (Pre-sort) and immediately after (Post-sort) MACS, and from the flow through fraction, were analyzed by FACS for enrichment of TRA-1-60 or SSEA4 positive stem cells. Two rounds of MACS were performed. Sort 1 was performed with TRA-1-60 or SSEA-4 antibodies in parallel and sort 2 was performed with either TRA-1-60 or SSEA4 antibody on cells previously sorted with TRA-1-60 antibody.
Fig 3.
Generation of iPSC pool by MACS from Sendai virus reprogrammed cells.
(A) Cell populations from before (Pre-sort) and immediately after (Post-sort) MACS, and from the flow through fraction, were analyzed by FACS for enrichment of TRA-1-60 or SSEA4 positive stem cells. Three rounds of MACS were performed. Sort 1 was performed with TRA-1-60 antibody; TRA-1-60 positive cells were then used for sort 2 with TRA-1-60 antibody again; and the resulting cells were used for sort 3 with SSEA4 antibody. (B) Phase contrast pictures of newly reprogrammed colonies before MACS and sorted cells cultured for 4 days after each round of antibody sorting (Post-sort). FACS plot of p4 Sendai virus reprogrammed cell pool after 3 rounds of sorting showing percentage of SSEA4+TRA-1-60+ cells (right panel).
Fig 4.
Characterization of MACS purified iPSC pool and clones.
(A) FACS analysis showing pool selected iPSCs (SVPool) stably express pluripotency markers TRA-1-60 and SSEA4 through long-term passaging up to p74, similar to three clonally derived iPSC lines (SV7, SV10, SV20) from the same donor. (B) Immunocytochemistry and (C) qRT-PCR analyses of mRNA levels from iPSCs showing SVPool and iPSC clones (p15–20) express similar levels of a panel of pluripotency markers tested. (D) SVPool and iPSC clones both show Sendai viral reprogramming vector clearance at p15–20 by RT-PCR analysis. (E) G-banded karyotyping was performed on SVPool and clones at p28–33 and all cell lines were found to be karyotypically normal. The data presented are chromosomal banding patterns of SVPool and one of the clones (SV10) for comparison.
Fig 5.
Comparison of in vitro differentiation potential of iPSC pool and clones.
(A) iPSC pool (SVPool) and clones (SV7, SV10, SV20) were induced to differentiate by embryoid body (EB) formation assay for 14 days. Multiple differentiations were performed for each cell line between p20–30. Total RNA isolated from the EBs was used for hPSC Scorecard analysis for tri-lineage differentiation potential. Scores for expression of pluripotency, ectoderm, mesoderm and endoderm lineage genes were calculated relative to a set of reference hESC and iPSC lines. Scores for the hESC line H9 was used for comparison. SVPool and all iPSC clones show similar differentiation potential. (B) SVPool and clones were induced to differentiate into hepatocyte-like cells. The iPSC line K3 was used as a positive control. Immunocytochemistry of day 21 differentiated hepatocytes show comparable expression of hepatocyte markers HNF4α and Albumin in all cell lines. (C) Taqman qRT-PCR analysis of mature hepatocyte markers show similar gene expression levels among hepatocytes derived from SVPool and iPSC clones. mRNA levels were expressed as fold changes relative to those of human primary hepatocytes. The iPSCs were induced for endothelial cell differentiation. At day 14 of differentiation, cells were sorted for CD31+CD144+ cells using FACS. A representative FACS plot of the percentage of CD31+CD144+ cells in the differentiation culture (D) and similar differentiation efficiencies (percentages) amongst SVPool and clones from two independent experiments (E) are shown.