Limits...
Genetic reporter analysis reveals an expandable reservoir of OCT4+ cells in adult skin.

Limbourg A, Schnabel S, Lozanovski VJ, Napp LC, Ha TC, Maetzig T, Bauersachs J, Naim HY, Schambach A, Limbourg FP - Cell Regen (Lond) (2014)

Bottom Line: However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms.Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP(+) epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response.Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany ; Integrated Research Center Transplantation (IFB-Tx), Hannover, Germany ; Department of Plastic, Hand and Reconstructive Surgery, Hannover, Germany.

ABSTRACT
The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4 (+) cells in postnatal mice. While the prevalence of GFP(+) cells in testis rapidly declined with age, the skin-resident GFP(+) population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP(+) epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.

No MeSH data available.


Related in: MedlinePlus

Characterization of GFP+cell populations in testis and skin in Oct4-Gfp (OG2) reporter mice. (A) Flow cytometry of cell suspensions from 1 wk old neonatal testis with compensation for autofluorescence by plotting FL1 against FL2. Dead cells are stained with propidium iodide (PI). (B) Timecourse analysis by flow cytometry of GFP + cells in testis at 0–2 wks, 3–4 wks, 6 wks and 24 wks of age. P vs 0–2 wks. (C) Representative FACS analysis and GFP+ gating of cell suspensions from skin. (D) Fluorescence microscopy of sorted GFP+ and GFP- cells for GFP, anti-OCT4 antibody staining (red) and nuclear dye (DAPI, blue). (E) GFP+ skin cells by FACS in different age groups (left), n = 36-45. Cyclic expansion of the GFP+ cell pool over time (right). n = 9-30. P vs 0.5 wks. (F) Immunostaining of hair follicle bulge region against smooth muscle α–actin (SMA) and GFP. Insert from same image in higher magnification. (G) Flow cytometry of GFP+ or GFP- gated cells from skin. CD34+ ITGα6hi and CD34+ ITGα6lo populations are indicated. (H) Representative flow cytometry profile of GFP+ gated cells stained with specific antibodies (spec. ab) or isotype control (isotype). (I) mRNA expression (2ΔCt) by quantitative real-time PCR of pluripotency factors from sorted skin cells or cultures of iPS and ES cells. n = 3-4. Scale bar: 20 um. Magnification: (D) 640, (F) 200, 640 (insert).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4230759&req=5

Fig1: Characterization of GFP+cell populations in testis and skin in Oct4-Gfp (OG2) reporter mice. (A) Flow cytometry of cell suspensions from 1 wk old neonatal testis with compensation for autofluorescence by plotting FL1 against FL2. Dead cells are stained with propidium iodide (PI). (B) Timecourse analysis by flow cytometry of GFP + cells in testis at 0–2 wks, 3–4 wks, 6 wks and 24 wks of age. P vs 0–2 wks. (C) Representative FACS analysis and GFP+ gating of cell suspensions from skin. (D) Fluorescence microscopy of sorted GFP+ and GFP- cells for GFP, anti-OCT4 antibody staining (red) and nuclear dye (DAPI, blue). (E) GFP+ skin cells by FACS in different age groups (left), n = 36-45. Cyclic expansion of the GFP+ cell pool over time (right). n = 9-30. P vs 0.5 wks. (F) Immunostaining of hair follicle bulge region against smooth muscle α–actin (SMA) and GFP. Insert from same image in higher magnification. (G) Flow cytometry of GFP+ or GFP- gated cells from skin. CD34+ ITGα6hi and CD34+ ITGα6lo populations are indicated. (H) Representative flow cytometry profile of GFP+ gated cells stained with specific antibodies (spec. ab) or isotype control (isotype). (I) mRNA expression (2ΔCt) by quantitative real-time PCR of pluripotency factors from sorted skin cells or cultures of iPS and ES cells. n = 3-4. Scale bar: 20 um. Magnification: (D) 640, (F) 200, 640 (insert).

Mentions: We verified the fidelity of the reporter by analyzing postnatal testis, since a subset of male spermatogonial stem cells (SSC) have been shown to express OCT4 and can give rise to pluripotent stem cells [12, 16–18]. FACS analysis of newborn mouse testis revealed a distinct cell population with specific GFP-fluorescence expressing OCT4 (Figure 1A, Additional file 1: Figure S1, Table 1). However, the prevalence of GFP+ cells in testis was highest in the early postnatal period but rapidly declined to permanently low levels with maturation ( ≥3-4 wks, Figure1B).Figure 1


Genetic reporter analysis reveals an expandable reservoir of OCT4+ cells in adult skin.

Limbourg A, Schnabel S, Lozanovski VJ, Napp LC, Ha TC, Maetzig T, Bauersachs J, Naim HY, Schambach A, Limbourg FP - Cell Regen (Lond) (2014)

Characterization of GFP+cell populations in testis and skin in Oct4-Gfp (OG2) reporter mice. (A) Flow cytometry of cell suspensions from 1 wk old neonatal testis with compensation for autofluorescence by plotting FL1 against FL2. Dead cells are stained with propidium iodide (PI). (B) Timecourse analysis by flow cytometry of GFP + cells in testis at 0–2 wks, 3–4 wks, 6 wks and 24 wks of age. P vs 0–2 wks. (C) Representative FACS analysis and GFP+ gating of cell suspensions from skin. (D) Fluorescence microscopy of sorted GFP+ and GFP- cells for GFP, anti-OCT4 antibody staining (red) and nuclear dye (DAPI, blue). (E) GFP+ skin cells by FACS in different age groups (left), n = 36-45. Cyclic expansion of the GFP+ cell pool over time (right). n = 9-30. P vs 0.5 wks. (F) Immunostaining of hair follicle bulge region against smooth muscle α–actin (SMA) and GFP. Insert from same image in higher magnification. (G) Flow cytometry of GFP+ or GFP- gated cells from skin. CD34+ ITGα6hi and CD34+ ITGα6lo populations are indicated. (H) Representative flow cytometry profile of GFP+ gated cells stained with specific antibodies (spec. ab) or isotype control (isotype). (I) mRNA expression (2ΔCt) by quantitative real-time PCR of pluripotency factors from sorted skin cells or cultures of iPS and ES cells. n = 3-4. Scale bar: 20 um. Magnification: (D) 640, (F) 200, 640 (insert).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230759&req=5

Fig1: Characterization of GFP+cell populations in testis and skin in Oct4-Gfp (OG2) reporter mice. (A) Flow cytometry of cell suspensions from 1 wk old neonatal testis with compensation for autofluorescence by plotting FL1 against FL2. Dead cells are stained with propidium iodide (PI). (B) Timecourse analysis by flow cytometry of GFP + cells in testis at 0–2 wks, 3–4 wks, 6 wks and 24 wks of age. P vs 0–2 wks. (C) Representative FACS analysis and GFP+ gating of cell suspensions from skin. (D) Fluorescence microscopy of sorted GFP+ and GFP- cells for GFP, anti-OCT4 antibody staining (red) and nuclear dye (DAPI, blue). (E) GFP+ skin cells by FACS in different age groups (left), n = 36-45. Cyclic expansion of the GFP+ cell pool over time (right). n = 9-30. P vs 0.5 wks. (F) Immunostaining of hair follicle bulge region against smooth muscle α–actin (SMA) and GFP. Insert from same image in higher magnification. (G) Flow cytometry of GFP+ or GFP- gated cells from skin. CD34+ ITGα6hi and CD34+ ITGα6lo populations are indicated. (H) Representative flow cytometry profile of GFP+ gated cells stained with specific antibodies (spec. ab) or isotype control (isotype). (I) mRNA expression (2ΔCt) by quantitative real-time PCR of pluripotency factors from sorted skin cells or cultures of iPS and ES cells. n = 3-4. Scale bar: 20 um. Magnification: (D) 640, (F) 200, 640 (insert).
Mentions: We verified the fidelity of the reporter by analyzing postnatal testis, since a subset of male spermatogonial stem cells (SSC) have been shown to express OCT4 and can give rise to pluripotent stem cells [12, 16–18]. FACS analysis of newborn mouse testis revealed a distinct cell population with specific GFP-fluorescence expressing OCT4 (Figure 1A, Additional file 1: Figure S1, Table 1). However, the prevalence of GFP+ cells in testis was highest in the early postnatal period but rapidly declined to permanently low levels with maturation ( ≥3-4 wks, Figure1B).Figure 1

Bottom Line: However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms.Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP(+) epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response.Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany ; Integrated Research Center Transplantation (IFB-Tx), Hannover, Germany ; Department of Plastic, Hand and Reconstructive Surgery, Hannover, Germany.

ABSTRACT
The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4 (+) cells in postnatal mice. While the prevalence of GFP(+) cells in testis rapidly declined with age, the skin-resident GFP(+) population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP(+) epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.

No MeSH data available.


Related in: MedlinePlus