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. 2023 Jun 7;24(12):9862.
doi: 10.3390/ijms24129862.

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans

Tetsuro Tamaki  1   2 , Toshiharu Natsume  1   2 , Akira Katoh  1   2 , Nobuyuki Nakajima  1   3 , Kosuke Saito  1   4 , Tsuyoshi Fukuzawa  1   5 , Masayoshi Otake  6 , Satoko Enya  6 , Akihisa Kangawa  6 , Takeshi Imai  1   7 , Miyu Tamaki  1   7 , Yoshiyasu Uchiyama  1   7
Affiliations

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans

Tetsuro Tamaki et al. Int J Mol Sci. .

Abstract

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

Keywords: GFP-transgenic pig; large animal experiment; micro-mini pig; multipotent stem cells; nerve-muscle regeneration; skeletal muscle.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sorting pattern of pig Sk-34 and Sk-DN cells. Sk-DN cells = P6 gate (Q1, orange dots), Sk-34 cells = P7 gate (Q2 and Q4, blue dots). Green dots in Q3 gate and others were excluded fraction mainly composed of debris and the other type of cells.
Figure 2
Figure 2
Expression of mRNAs specifically for the skeletal muscle (green; 1~9), peripheral nerve (pink; 10~16) and vascular (blue; 17~20) cell lineages of the Sk-DN and Sk-34 cells. Grey = CD34 (21). Black = GAPDH (housekeeping gene; 22). Expression was evaluated between 0 to 3 based on the value of GAPDH, and 3 is the highest. Samples were obtained from the non-GFP-Tg-MMP (n = 4). In this case, the total RNA extracts were preliminary mixed (averaged) and analyzed.
Figure 3
Figure 3
Percent distribution of Px7+ and MyoD+ cells in the Sk-34 and Sk-DN cells based on total cells. The non-GFP-Tg-MMP was used in this analysis (n = 4), and 2–3 cytospin preparations/animal were counted. Values are expressed mean ± S.E. * p < 0.05.
Figure 4
Figure 4
In vivo differentiation capacity of Sk-DN cells. Samples were obtained from the mouse TA injury model 5-weeks after operation. (A): anti-Skeletal muscle actin (red staining), (B–D): anti-Laminin staining (red staining). Inset 1 and 2 (dotted squares) in panel (B) correspond to the part as panel (C,D). Arrows in (C,D) shows typical small fiber having laminin. Dotted oblong line point to the possible involvement of green cells as satellite cells. Blue staining = DAPI. Bars = 100 μm.
Figure 5
Figure 5
In vivo differentiation capacity of Sk-34 cells. The results of skeletal muscle and vascular lineage are from the mouse TA damage model, and peripheral nerve lineage are from the rat sciatic nerve damage model. In the immunohistochemical photographs (A,B,E,F), left column = GFP + cells/tissues; center = reactions for each antibody; right column = merge image. Arrows show typical portions of double reactions with GFP and red reactions for each antibody (yellow colors). Blue staining = DAPI. (C,D): Immunoelectron microscopy. Dark portions are DAB+ reactions for anti-GFP. PN = perineurium, MN = myelinated nerve, PC = perineurial cell, Cp = capillary, EC = endoneurial cell, EN = endoneurium. Bars = 30 μm.
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