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Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.

Byrne JA, Nguyen HN, Reijo Pera RA - PLoS ONE (2009)

Bottom Line: Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming.Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, California, United States of America.

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.

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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.
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pone-0007118-g002: 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.

Mentions: We next examined whether the expression of SSEA3 in a subset of fibroblasts was specific to HUF1 or a more general observation. Eight additional primary adult human fibroblast lines were derived from skin biopsies and immunoassayed. We observed that all eight lines contained a subpopulation of cells that were positive for SSEA3 (Figure 2A). Fluorescence activated cell sorting (FACS) analysis of HUF1 cells stained with the SSEA3/488 antibody complex, revealed a larger subpopulation of cells with little or no SSEA3 expression and a smaller subpopulation with detectable SSEA3 expression (Figure 2B). Subsequently, we isolated (through FACS) and cultured the top 10% and bottom 10% of the SSEA3/488 fluorescing cells as our SSEA3-positive and negative populations respectively (Figure 2B). Immunofluorescence analysis of the two populations, following overnight adherence to exclude analysis of non-viable cells, revealed that >97% of the SSEA3-positive population expressed detectable SSEA3/488 fluorescence and 0% of the SSEA3-negative population expressed detectable SSEA3/488 fluorescence (Figure 2C), demonstrating that the fluorescence activated cell sorting process can purify viable subpopulations of cells from a heterogeneous somatic population. These subpopulations were then used for reprogramming to iPSCs.


Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.

Byrne JA, Nguyen HN, Reijo Pera RA - PLoS ONE (2009)

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.
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Related In: Results  -  Collection

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pone-0007118-g002: 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.
Mentions: We next examined whether the expression of SSEA3 in a subset of fibroblasts was specific to HUF1 or a more general observation. Eight additional primary adult human fibroblast lines were derived from skin biopsies and immunoassayed. We observed that all eight lines contained a subpopulation of cells that were positive for SSEA3 (Figure 2A). Fluorescence activated cell sorting (FACS) analysis of HUF1 cells stained with the SSEA3/488 antibody complex, revealed a larger subpopulation of cells with little or no SSEA3 expression and a smaller subpopulation with detectable SSEA3 expression (Figure 2B). Subsequently, we isolated (through FACS) and cultured the top 10% and bottom 10% of the SSEA3/488 fluorescing cells as our SSEA3-positive and negative populations respectively (Figure 2B). Immunofluorescence analysis of the two populations, following overnight adherence to exclude analysis of non-viable cells, revealed that >97% of the SSEA3-positive population expressed detectable SSEA3/488 fluorescence and 0% of the SSEA3-negative population expressed detectable SSEA3/488 fluorescence (Figure 2C), demonstrating that the fluorescence activated cell sorting process can purify viable subpopulations of cells from a heterogeneous somatic population. These subpopulations were then used for reprogramming to iPSCs.

Bottom Line: Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming.Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus