Mesenchymal progenitor cells derived from traumatized human muscle

W. M. Jackson (1), A. B. Aragon (1,2), F. Djouad (1), Y. Song (1), S. M. Koehler (1), L. J. Nesti (1,2) and R. S. Tuan (1) (1) - Cartilage Biology and Orthopaedic Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA
(2) - Integrated Department of Orthopaedics and Rehabilitation, Walter Reed Army Medical Center, Washington, DC, USA

Published online 23 January 2009.
Journal of Tissue Engineering and Regenerative Medicine

Background:

Mesenchymal stem cells (MSCs) show great promise in regenerative medicine and tissue engineering since they can differentiate into several cell lineages (osteoblasts, adipocytes, and chondrocytes) with additional specific protocols that can induce MSCs to differentiate into several others (myoblasts, cardiomycocytes, heptocytes, and neurons). The clinical applications of these cells are also not hindered by ethical concerns since these cells are derived from adult tissues. However, the difficulty with MSCs is availability. Most, if not all, autologous MSCs that could be used for clinical applications are limited by how invasive the procedure is to collect these cells. Thus, any novel expansion of sources for MSCs is required for an expansion of these cells in clinical use.

One area that could readily benefit from an available source of MSCs is that of orthopaedic traumatology. Most orthopaedic extremity traumas usually require circumferential debridement of contaminated and devitalized tissue. The excision of this tissue result in a loss of a potential source of MSCs. Yet, research has shown that MSCs are not usually found in muscle tissue compared to that of bone tissue. However, this paper indicates that there is a substantial population of mesenchymal progenitor cells (MPCs) that reside in muscle tissue, particularly the traumatized muscle tissue of patients with extremity trauma. Thus, the goal of the paper is to characterize these MPCs in hope that there is a significant similarity in cell morphology, proliferation capacity, cell surface epitope profile, and the differentiation profile of MPCs derived from traumatized muscle tissue compared to that of bone marrow derived MSCs.

Summary:

The research was done primarily at Walter Reed Army Medical Center in Washington, DC. In order to harvest the MPCs, twenty patients with orthopaedic trauma and sustained extensive soft tissue extremity wounds had uncontaminated non-necrotic muscle collected. The samples were then digested and cultured. In order to compare the relative similarities or differences in MPCs and MSCs, bone marrow was harvested from four individuals undergoing hip replacement. These cells were cultured under standard, published protocols. After both groups of samples had proliferated, five assays were employed to compare the overall profiles of MSCs and MPCs. The study concluded that MPCs derived from traumatized muscle were functionally similar to MSCs derived from bone marrow.

Methods:

Note: This is a broad overview of the methods employed. For a more specific protocol, consult the paper.

The profile of a patient with MPCs harvested:

• Age: 24.4 +/- 5.3 years old
• Male
• Orthopaedic trauma & sustained extensive soft tissue extremity wounds
• Arrival time from the field: 3-7 days
• Collection of Tissue: 2-3 days
• n = 20 samples (16 patients, 4 patients had injuries on two extremities)

Approximately 200 μg of tissue was dissected, removing contamination of granulation, adipose or fibrous tissues. The muscle sample was then cultured in DMEM and minced before being digested via 0.5 mg/ml collaganase type 2. This was then pelleted and then resuspeneded in DMEM with 10% FBS and 5 units/mL pen/strep and fungizone (PSF). The sample was then cultured for 2 hours before washing in HBSS (Hank's buffered saline solution). Finally, the sample was incubated in DMEM with 10% FBS and 1 unit/mL PSF until ~85% confluent.

Whereas, the profile of a patient with MSCs harvested:

• Age: 63.8 +/- 8.5 years
• 50% Male , 50% Female
• n = 4

In this case, whole bone segments were placed in dishes and marrow was isolated from those pieces. The marrow was then centrifuged before being cultured in DMEM with 10% FBS and 1 unit/mL PSF until ~85% confluent.

The five assays used were:

1. MTT Assay
2. Flow Cytometry:
3. Differentiation Assays:
4. Histology & Immunohistochemistry:
5. RT-PCR:

Results:

Note: All Figure Captions are from Jackson, et al.

The overall results indicated a morphological similarity between both samples and that the MTT assay, testing for proliferation rates, did not indicate any significance between the two. This is shown in Figure 1. As to the overall surface epitopes, the MPCs were positive for characteristic MSC markers (CD44, CD49e, CD73, CD90, and CD105) whilst negative for HSCs markers (CD14, CD31, CD34, and CD45).

Whereas, the differentiation assays demonstrated that MPCs from traumatized muscle could differentiate into osteblasts and adipocytes. This was shown from increased alkaline phosphatase activity compared to MPCs cultured in GM whilst producing a mineralized matrix as seen from staining with Alizarin red. MPCs cultured in adipogenic medium also showed adipogenic characteristics; mostly that of intracellular lipid droplets and upregulation of PPARG2 (master regulator of adipogenesis). The same could be said of chondrocytes, however, myogenic culture conditions had no effect on either MPCs or MSCs cultures.

Figure 1. Morphology of MPCs derived from traumatized muscle (A,C) and MSCs derived from bone marrow (B, D). The MPCs resembled the long, spindle-shaped MSCs when they are isolated (A, B) and when they were near-confluent (C, D).
Scale bar = 25 μm. (#) The proliferation of muscle-derived MPCs and bone marrow-derived MSCs measured using the MTT reduction assay. The amount of MTT reduction product was not significantly different between the MPCs and MSCs on days 3 or 7, suggesting that the proliferation rates of the two cell types were the same. Data from cells derived from three different patients (mean +/- SD)







Figure 2. Flow cytometric analysis of surface epitope profiles of traumatized muscle-derived MPCs and bone marrow-derived MSCs. (A) The traumatized muscle=derived MPCs were positive for CD73, CD90 and CD105 and negative for CD14, CD34 and CD45. The black lines represent the fluorescence intensity of cells stained with the indicated antibodies and the grey lines represent the negative control cells, which were stained with a non-immunoreactive isotype control antibody. (B) There were no significant differences in the normalized fluorescence intensities of MPCs or bone marrow-derived MSCs that were stained with each antibody. The fluorescence intensities were normalized to the negative control cells. Data from cells derived from three different patients (mean +/- SD)

Figure 3. Osteogenic differentiation of MPCs derived from traumatized muscle. MPCs cultured in osteogenic medium exhibited increased ALP activity (B) compared to MPCs cultured in growth medium (A) and produced a mineralized matrix stained with Alizarin red (E). MPCs cultured in grwoth medium exhibited little Alizarin red staining (D). Positive controls: bone marrow-derived MSCs cultured in osteogenic medium -ALP activity (C) and Alizarin red staining (F). Scale bar = 5 mm. (G) MPCs cultured in osteogenic medium (medium O) upregulated the expression of osteoblast-specific genes [CBFA1/RUNX, ALP (alkaline phosphatase)0 and osteocalcin] compared to cells cultured in growth medium (medium G). This gene expression profile was similar to that of bone marrow derived MSCs, although the expression of the osteoblast-specific genes was also seen under growth conditions in the MSCs.





Figure 4. Adipogenic differentiation of MPCs derived from traumatized muscle. MPCs derived from traumatized muscle and cultured in adipogenic medium formed intracellular lipid droplets that were stained using oil red O (B). MPCs cultured in growthmedium did not produce any oil droplets (A). (C) Positive

control: bone marrow-derived MSCs cultured in adipogenic medium with oil red O staining. Scale bar = 50 μm. (D) MPCs cultured in adipogenic medium (medium A) upregulated
the expression of adipocyte-specific genes [PPARG2, LPL (lipoprotein lipase) and FABP4 (fatty acid binding protein 4)] compared to cells cultured in growth medium (medium
G). This gene expression profile was characteristic of bone
marrow-derived MSCs

Figure 5. MPCs derived from traumatized muscle and cultured
under chondrogenic pellet conditions (A–C, G, H) compared to
bone marrow-derived cells cultured under identical conditions
(D–F, I, J). The gross morphology of the chondrogenic pellets
was similar for both cell types (A, D; scale bar = 500 μm). Alcian
blue-stained sections of the pellets were imaged at ×4 (B, E; scale
bar = 250 μm) and ×10 (C, F; scale bar = 200 μm) showing the
presence of a sulfated proteoglycan-rich extracellular matrix.
Pellets of both cell types stained positively for collagen
type II (G, I) and aggrecan (H, J; scale bar = 250 μm).
(K) The expression of chondrogenic genes [SOX9, COL2A1, AGC
(aggrecan), COMP and COL10A1] at days 7 and 21 in traumatized
muscle-derived MPCs cultured under chondrogenic conditions
was characteristic of bone marrow-derived MSCs undergoing
chondrogenic differentiation



Critique:

The point of this study was to find a new source of defined cells that were characteristically similar to mesenchymal stem cells, in this case, mesenchymal progenitor cells from traumatized muscle tissue. To an extent, the researchers seem to have succeeded in finding their cell source. Unfortunately, there are some caveats that are concerning. First, the study does not take into account the effects of trauma on the harvested MPCs. There is no known studies if MPCs reside in tissue that are not damaged to the extent the muscle tissue were damaged in the cases examined. The question of whether or not progenitor activation may be a causal reaction to inflammation and would healing factors is never answered. The second caveat is the maturity of these cell passages. The paper itself admits that the experiments conducted on the cells were done when the cells were only at around passage 10-12 and in some instances at around passage 8. This is in no way a clear indication that the MPCs themselves are able to expansively proliferate at later passages in vitro without any difficulties.