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Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin.

Mallon BS, Hamilton RS, Kozhich OA, Johnson KR, Fann YC, Rao MS, Robey PG - Stem Cell Res (2013)

Bottom Line: We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions.Analysis of the global methylation pattern also showed no significant difference between the two PSC populations.However, these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level.

View Article: PubMed Central - PubMed

Affiliation: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: mallonb@mail.nih.gov.

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In vitro pluripotency assays for iPSC line, H1.NPC-i3. A & B) Endoderm differentiation – A) Directed differentiation generates cells positive for the hepatocyte markers, albumin (green) and HNF4α (red) as well as for alpha-fetoprotein (AFP) in B). C) Ectoderm differentiation – Neurons derived from H1.NPC-i3 are immunopositive for TuJ1 (green) with many MAP-2 positive cells (red). D) Mesoderm differentiation-cardiomyocytes are immunopositive for myosin heavy chain (green) and Troponin T (red). Note: Beating cardiomyocytes, indicative of mesoderm differentiation can also be viewed in Supplementary Video 1. Scale bar = 100 μm.
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Figure 2: In vitro pluripotency assays for iPSC line, H1.NPC-i3. A & B) Endoderm differentiation – A) Directed differentiation generates cells positive for the hepatocyte markers, albumin (green) and HNF4α (red) as well as for alpha-fetoprotein (AFP) in B). C) Ectoderm differentiation – Neurons derived from H1.NPC-i3 are immunopositive for TuJ1 (green) with many MAP-2 positive cells (red). D) Mesoderm differentiation-cardiomyocytes are immunopositive for myosin heavy chain (green) and Troponin T (red). Note: Beating cardiomyocytes, indicative of mesoderm differentiation can also be viewed in Supplementary Video 1. Scale bar = 100 μm.

Mentions: At p5, H1.NPCs were reprogrammed using the Millipore StemCCA lentiviral kit and plated out on MEF feeders in hESC medium. A total of 24 colonies were individually isolated directly from the feeder well and expanded on feeders. Remaining colonies were collected by collagenase digestion and replated on Matrigel. From this plate a further 12 colonies were individually isolated and expanded on Matrigel. Of these isolated colonies, 3 from each culture condition were analyzed by FACS for SSEA4, Tra-1-60 and NCAM. All cell lines were found to express SSEA4 and Tra-1-60, were immunonegative for SSEA1 and NCAM, as determined by FACS analysis, and were immunopositive for Oct4 (Fig. 1B and Supplementary Fig. 2). The three feeder-based iPSC lines, designated H1.NPC-i1, -i2 and -i3, were confirmed by STR analysis to have an identical genetic profile to the parent H1 line and had a normal karyotype at p14, p16 and p18 respectively (Supplementary Fig. 3). Each of these lines also exhibited a PCR product indicative of the presence of reprogramming viral sequences not present in the parent lines or neural precursor intermediate (Supplemental Fig. 4). By gene expression microarray analysis, the iPSC lines were shown to express the pluripotency genes, NANOG and OCT4, as well as telomere reverse transcriptase (TERT), at similar levels to hESC (Fig. 1C). These levels were reduced in the NPC population. SOX2, which is also present in neural precursors, is highly expressed in all cells (Fig. 1C). Two lines, H1.NPC-i1 and H1.NPC-i3, were tested for in vitro differentiation potential and were successfully directed to differentiate into cells representative of the 3 germ lineages. This is represented by Fig. 2 showing data for H1.NPC-i3 differentiation. Endoderm differentiation is demonstrated by immunoreactivity to AFP (Fig. 2A) as well as to HNF4α and albumin (Fig. 2B; hepatocytes), ectoderm differentiation by nestin and TuJ1 immunoreactivity (Fig. 2C; neurons) and mesoderm differentiation by myosin heavy chain β and troponin T immunoreactivity (Fig. 2D; cardiomyocytes) as well as by patches of spontaneously beating cells (Supplementary Video 1).


Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin.

Mallon BS, Hamilton RS, Kozhich OA, Johnson KR, Fann YC, Rao MS, Robey PG - Stem Cell Res (2013)

In vitro pluripotency assays for iPSC line, H1.NPC-i3. A & B) Endoderm differentiation – A) Directed differentiation generates cells positive for the hepatocyte markers, albumin (green) and HNF4α (red) as well as for alpha-fetoprotein (AFP) in B). C) Ectoderm differentiation – Neurons derived from H1.NPC-i3 are immunopositive for TuJ1 (green) with many MAP-2 positive cells (red). D) Mesoderm differentiation-cardiomyocytes are immunopositive for myosin heavy chain (green) and Troponin T (red). Note: Beating cardiomyocytes, indicative of mesoderm differentiation can also be viewed in Supplementary Video 1. Scale bar = 100 μm.
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Related In: Results  -  Collection

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Figure 2: In vitro pluripotency assays for iPSC line, H1.NPC-i3. A & B) Endoderm differentiation – A) Directed differentiation generates cells positive for the hepatocyte markers, albumin (green) and HNF4α (red) as well as for alpha-fetoprotein (AFP) in B). C) Ectoderm differentiation – Neurons derived from H1.NPC-i3 are immunopositive for TuJ1 (green) with many MAP-2 positive cells (red). D) Mesoderm differentiation-cardiomyocytes are immunopositive for myosin heavy chain (green) and Troponin T (red). Note: Beating cardiomyocytes, indicative of mesoderm differentiation can also be viewed in Supplementary Video 1. Scale bar = 100 μm.
Mentions: At p5, H1.NPCs were reprogrammed using the Millipore StemCCA lentiviral kit and plated out on MEF feeders in hESC medium. A total of 24 colonies were individually isolated directly from the feeder well and expanded on feeders. Remaining colonies were collected by collagenase digestion and replated on Matrigel. From this plate a further 12 colonies were individually isolated and expanded on Matrigel. Of these isolated colonies, 3 from each culture condition were analyzed by FACS for SSEA4, Tra-1-60 and NCAM. All cell lines were found to express SSEA4 and Tra-1-60, were immunonegative for SSEA1 and NCAM, as determined by FACS analysis, and were immunopositive for Oct4 (Fig. 1B and Supplementary Fig. 2). The three feeder-based iPSC lines, designated H1.NPC-i1, -i2 and -i3, were confirmed by STR analysis to have an identical genetic profile to the parent H1 line and had a normal karyotype at p14, p16 and p18 respectively (Supplementary Fig. 3). Each of these lines also exhibited a PCR product indicative of the presence of reprogramming viral sequences not present in the parent lines or neural precursor intermediate (Supplemental Fig. 4). By gene expression microarray analysis, the iPSC lines were shown to express the pluripotency genes, NANOG and OCT4, as well as telomere reverse transcriptase (TERT), at similar levels to hESC (Fig. 1C). These levels were reduced in the NPC population. SOX2, which is also present in neural precursors, is highly expressed in all cells (Fig. 1C). Two lines, H1.NPC-i1 and H1.NPC-i3, were tested for in vitro differentiation potential and were successfully directed to differentiate into cells representative of the 3 germ lineages. This is represented by Fig. 2 showing data for H1.NPC-i3 differentiation. Endoderm differentiation is demonstrated by immunoreactivity to AFP (Fig. 2A) as well as to HNF4α and albumin (Fig. 2B; hepatocytes), ectoderm differentiation by nestin and TuJ1 immunoreactivity (Fig. 2C; neurons) and mesoderm differentiation by myosin heavy chain β and troponin T immunoreactivity (Fig. 2D; cardiomyocytes) as well as by patches of spontaneously beating cells (Supplementary Video 1).

Bottom Line: We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions.Analysis of the global methylation pattern also showed no significant difference between the two PSC populations.However, these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level.

View Article: PubMed Central - PubMed

Affiliation: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: mallonb@mail.nih.gov.

Show MeSH