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. 2011 Nov;15(11):2377-88.
doi: 10.1111/j.1582-4934.2010.01225.x.

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Wesley M Jackson  1 , Thomas P Lozito, Farida Djouad, Nastaran Z Kuhn, Leon J Nesti, Rocky S Tuan
Affiliations

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Wesley M Jackson et al. J Cell Mol Med. 2011 Nov.

Abstract

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.

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Figures

Fig 1
Fig 1
Morphology, proliferation and metabolism of traumatized muscle derived MPCs. (A) Representative sections of the traumatized muscle demonstrating the extent of tissue damage compared to control muscle. Bar = 200 μm; haematoxylin and eosin. (B) MPC morphology was similar to that of bone marrow derived MSCs under phase contrast microscopy. Bar = 50 μm. (C) The proliferation of MPCs and MSCs was assayed on the basis of dsDNA, and no significant differences were detected. (D) Metabolic activity was measured on the basis of the chemical reduction capacity, and 5 days after seeding, a significant difference was detected between MPCs and MSCs. *P < 0.05, Student’s t-tests with n= 4.
Fig 2
Fig 2
Cell surface epitope profile of traumatized muscle derived MPCs. (A) MPC cell surface marker expression (black dots) compared to isotype control (grey dots) were compared using flow cytometry. All antibodies were PE conjugated (red), and the percentage of events with elevated FL-2 fluorescence is indicated in each panel (mean ± S.D. for n= 4). (B) The fluorescence intensity of positive surface markers was compared between MPCs and bone marrow derived MSCs. *P < 0.05, Student’s t-tests with n= 4, †the fluorescence intensity of CD90 is 10× greater than the other surface markers and is depicted using the right axis.
Fig 3
Fig 3
Trilineage differentiation of traumatized muscle derived MPCs. (A) Histological evidence of differentiation in MPCs compared to bone marrow derived MSCs grown in GM, OM, AM and chondrogenic induction medium with (TGF-β+) and without TGF-β3 (TGF-β–). Scale bars: ALP, alizarin red and oil red O: 100 μm. Gross pellet: 500 μm. Whole pellet section: 400 μm. 10× pellet: 200 μm. (B–F) Quantitative assays of differentiation: (B) ALP activity assay for osteogenic differentiation, (C) oil red O inclusion assay for adipogenesis, (D) pellet size, (E) total sGAG production and (F) normalized sGAG production. a, b and c: P < 0.05 with one-way anova and Student-Newman-Keuls (SNK) multiple comparison tests and n= 4, *P < 0.05, Student’s t-tests with n= 4.
Fig 4
Fig 4
qPCR array for MSC genes. (A) Comparison of the gene expression profiles for traumatized muscle derived MPCs and bone marrow derived MSCs. Genes with 4-fold differential expression (indicated by dashed lines) are labelled and select genes are listed in the adjacent table. (B) A volcano plot comparing the fold-difference in cytokine gene expression (x-axis; vertical dashed lines indicating 4-fold differential expression) to the statistical significance (y-axis; horizontal dashed line indicating P < 0.05). Bar graphs comparing: (C) specific MSC Markers (THY1= CD90, ENG= CD105 and VCAM1= CD106) and (D) baseline expression of master regulators associated with trilineage differentiation potential. *P < 0.05, Student’s t-tests with n= 3.
Fig 5
Fig 5
Immunosuppression by traumatized muscle derived MPCs. (A) Comparison of immunoregulatory gene expression between MPCs and bone marrow derived MSCs. (B) T-cell proliferation in a mixed lymphocyte reaction plotted as a percentage of total proliferation in response to an antigen without modulation by MPCs or MSCs. *P < 0.05, Student’s t-tests and n= 3.
Fig 6
Fig 6
Vascular maintenance associated with traumatized muscle derived MPCs. (A) Comparison of angiogenic gene expression between MPCs and bone marrow derived MSCs. (B) Western blots to verify VEGF protein expression in the cell supernatants at days 1, 2 and 4. (C) Proliferation of microvascular endothelial cells at 48 and 72 hr time-points. The cells were cultured in media that had been conditioned by MPCs, MSCs or no cells for 4 days prior to plating. a, b, c and d: P < 0.05, one-way anova and SNK multiple comparisons and n= 4.
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