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. 2009 Sep 23;4(9):e7118.
doi: 10.1371/journal.pone.0007118.

Enhanced Generation of Induced Pluripotent Stem Cells From a Subpopulation of Human Fibroblasts

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Free PMC article

Enhanced Generation of Induced Pluripotent Stem Cells From a Subpopulation of Human Fibroblasts

James A Byrne et al. PLoS One. .
Free PMC article

Abstract

Background: The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming.

Methodology/principal findings: Here, we report identification and isolation of a subpopulation of human dermal fibroblasts that express the pluripotency marker stage specific embryonic antigen 3 (SSEA3). Fibroblasts that expressed SSEA3 demonstrated an enhanced iPSC generation efficiency, while no iPSC derivation was obtained from the fibroblasts that did not express SSEA3. Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the differential reprogramming.

Conclusions/significance: To our knowledge, this study is the first to identify a pluripotency marker in a heterogeneous population of human dermal fibroblasts, to isolate a subpopulation of cells that have a significantly increased propensity to reprogram to pluripotency and to identify a possible mechanism to explain this differential reprogramming. This discovery provides a method to significantly increase the efficiency of reprogramming, enhancing the feasibility of the potential applications based on this technology, and a tool for basic research studies to understand the underlying reprogramming mechanisms.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of SSEA3 from primary human dermal fibroblasts.
(A–B) Primary adult human fibroblast line HUF1 (A) Phase contrast image (B) Immunocytochemical detection of SSEA3 expression (green). (C–D) Immunofluorescence staining for (C) TRA-1-60 and (D) TRA-1-81 on HUF1 cells. (E) Overlay of SSEA3 expression on phase contrast image of HUF1 cells. (F) Confocal section through primary human fibroblast (HUF1) cell demonstrating SSEA3/488 detection primarily from the cell membrane in addition to localized peri-nuclear detection. (G) SSEA3/488 detection on H9 human embryonic stem cells. (H) 488 secondary antibody only negative control staining of HUF1 cells. (C–H) DAPI staining in blue. Scale bars represent 100 microns.
Figure 2
Figure 2. FACS analysis and isolation of SSEA3-positive and SSEA3-negative primary adult human fibroblasts.
(A) Immunocytochemical analysis for SSEA3 expression in eight additional primary adult dermal human fibroblast (HUF) lines. DAPI staining in blue. (B) Histogram of FACS analyzed HUF1 cells following live binding of SSEA3/488 antibody complex and gating for SSEA3-positive (top 10%) and SSEA3-negative (bottom 10%) populations. (C) Detection of SSEA3/488 fluorescence signal in FACS sorted SSEA3-positive and SSEA3-negative populations following overnight adherence. (A & C) SSEA3 staining in green. Scale bars represent 100 microns.
Figure 3
Figure 3. Characterization of unsorted HUF1 derived induced pluripotent stem cells (HiPS-1 control).
(A) Expression of pluripotency markers from iPSCs (HiPSC-1 control) generated following retroviral transduction of unsorted HUF1 cells. DAPI staining in blue. Scale bar represents 100 microns. (B) SKY karyotype analysis of the HiPS-1 control line. (C) Histological analysis of teratoma derived from HiPS-1 control line.
Figure 4
Figure 4. Morphology of colonies and lines following retroviral transduction of HUF1 cells.
(A) Large background colony with no ESC-like attributes. (B) ESC-like iPSC colony. (C) Morphology of SSEA3-selected lines following derivation. (A–C) Scale bar represents 100 microns.
Figure 5
Figure 5. Characterization of SSEA3-selected HiPSCs.
(A) Expression of pluripotency markers on H9 ESCs and SSEA3-selected HiPSC lines. Alkaline phosphatase (AP) staining in dark red/purple. DAPI stained images are inset in blue. (B) Spectral karyotype (SKY) of SSEA3-selected HiPS-2C line. (C) Histological analysis of teratoma derived from SSEA3-selected HiPS-2C line. (A & C) Scale bar represents 100 microns.
Figure 6
Figure 6. Transcriptional analysis of primary dermal fibroblast subpopulations with differential SSEA3 expression.
Relative expression of Nanog, Sall4, hTert and Gapdh from three subpopulations of HUF1 cells: SSEA3-negative cells (representing the bottom 10% for SSEA3 expression/detection), SSEA3 intermediate cells (representing the intermediate 80% of cells between the top and bottom 10% for SSEA3 expression/detection) and SSEA3-positive cells (representing the top 10% for SSEA3 expression/detection). Three biological replicates were analyzed for each sample. The relative gene expression value represents the level of gene expression for each sample compared to the overall average for that gene across the three subpopulations.

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