Neuro-muscular differentiation of adult porcine skin derived stem cell-like cells

Abstract

Background: Due to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time.

Methodology/principal findings: pSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions.

Conclusions/significance: Adult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes).

Figure 1. Growth characteristics of pSSCs in vitro.

(A) Proliferation of pSSCs: Given is the CPD (cumulative population doubling) of pSSCs as function of days in culture. Data are expressed as means ± SEM from three independent pSSC strains. (B) Average CFU efficiency of one representative pSSCs strain after 22 CPD. Mean of three experiments ± SEM. (C) Image of cell colonies (stained with trypan-blue) formed after 11 days seeded with 10 cells per cm² growth surface. Scale bar: 1 cm. (D, E) Morphological appearance of pSSCs. Scale bars: (D) 200 µm, (E) 100 µm.

Figure 2. Expression of stem cell related genes and translated proteins.

(A) RT-PCR analysis of stem cell related genes. Line 1–3: results of three independent cell strains, line 4: negative control (-RT) from one representative cell strain. (B) Flow cytometric analysis of cell surface markers CD9, CD29, CD44, CD90, CD105, and nestin. Data are expressed as means ± SEM from three independent pSSCs strains after 22 CPD. (C) Immunocytochemistry revealed a subpopulation of pSSCs expressing nestin. (D) Double staining indicates the coexpression of nestin and fibronectin. Scale bars: 50 µm.

Figure 3. Neuro-muscular differentiation potential of pSSCs.

(A) After 30 days of induced neuronal differentiation one cell strain developed a structured cell network of linked bodies. (B–D) Cells show morphologies similar of neuronal cells (white arrows) and smooth muscle cells (black arrows). Scale bars: (A, B) 500 µm, (C) 50 µm, (D) 100 µm. (C) Represents the marked region in Fig. 3B. (E–G) Immunocytochemistry revealed the expression β-III-tubulin and GFAP in the centre and some surrounding cells of the bodies, whereas α-SMA is exclusively expressed in the outer regions of the bodies and some surrounding cells. Scale bars: 50 µm. (H, I) β-III-tubulin is expressed in a subpopulation of cells that are morphological consistent with neurons. (J, K) Other subpopulations of differentiated pSSCs expressed the neuronal markers GFAP and neurofilament M. (L, M) Subpopulations with a morphology consistent of smooth muscle cells expressed the smooth muscle marker α-SMA. Scale bars: 50 µm.

Figure 4. Differentiation-related regulation of proteins.

Expression of the neuronal and muscular lineage markers nestin, beta-III-tubulin, GFAP, and α-SMA before differentiation (white bars) and after differentiation (grey bars) revealed by flow cytometry. Data are expressed as means ± SEM from three independent cell strains.

Figure 5. Induced adipogenic differentiation of pSSCs.

(A) The adipogenic lineage differentiation is shown by bright field pictures of positive Oil Red O stained lipid vacuoles within the cytoplasm of the pSSCs after 30 days of induced differentiation. (B) Oil red O staining of pSSCs cultured in control medium. Scale bars: 50 µm. (C) Leptin expression shown by RT-PCR in adipogenic differentiated and undifferentiated pSSC cultures. Hypoxanthin-guanin phosphoribosyltransferase (HPRT) was used as an internal control for each PCR.