Chondrogenic progenitor cells isolated from the fascia of skeletal muscle

doi:10.3969/j.issn.2095-4344.0198

Abstract

Abstract:

BACKGROUND: Muscle derived cells in the skeletal muscle have the ability to differentiate into chondrocytes under the induction of bone morphogenic protein 4 and transforming growth factor β3. However, due to the complexity of the skeletal muscle (consisting of muscle fiber, fascia, blood vessel and nerves), the origin of cells has not been identified.
OBJECTIVE: To observe the potential for chondrogenic differentiation of fascia-derived cells (FDCs) in order to clarify the chondrogenic ability of the cells derived from the skeletal muscle.
METHODS: The fascia was isolated from the rat gluteus maximus, and was then cultured in chondrogenic medium containing bone morphogenic protein 4 and transforming growth factor β3. Samples were harvested on day 14. The chondrogenic differentiation of FDCs was identified by alcian blue, hematoxylin-eosin and safranin O stainings. Cell markers were investigated by immunostaining using the antibodies for desmin, CD34, vimentin, vWF and a-SMA. Immunostaining was used to assay the chondrogenic potential of FDCs cultured with L6 rat myoblasts at varying ratios (1:0, 4:1; 1:1, 1:4 and 0:1).
RESULTS AND CONCLUSION: FDCs harvested from the skeletal muscle displayed chondrogenic differentiation and formed cartilaginous tissue when cultured for 14 days in chondrogenic medium after alcian blue and safranin O stainings. Cells isolated from the fascia were negative for desmin, CD34, vWF and a-SMA, but over 90% of the cells were positive for vimentin. Mixed FDC and L6 myoblast pellets showed chondrogenic potential decreased with the increasing ratio of L6 myoblasts. From the analysis of cell surface marker of FDCs in the skeletal muscle, immunostaining, and the comparison with L6 rat myoblasts, it shows that FDCs are the seed cells with chondrogenic potential in the skeletal muscle.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Muscle, Skeletal, Fascia, Chondrocytes, Tissue Engineering

CLC Number:

R318

Xiang Yong, Li Guang-heng. Chondrogenic progenitor cells isolated from the fascia of skeletal muscle[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(12): 1823-1828.

Figures/Tables 1

2.1 臀大肌筋膜组织染色和其软骨细胞分化能力的分析取Fischer 344大鼠臀大肌肌筋膜进行活检，苏木精-伊红染色结果显示，筋膜组织主要是由包裹骨骼肌的纤维和结缔组织组成(图1)；筋膜组织块在软骨细胞培养基中培养，在软骨形成培养基中培养14 d之后，番红O和阿尔新蓝染色后可观察到软骨细胞的典型圆形形态和典型的细胞外基质，表明有软骨组织形成(图2)。 2.2 细胞表面标记物分析筋膜组织分离细胞的免疫组织化学染色显示，细胞核被染成蓝色，结蛋白被染成红色；在软骨形成基质培养之后，筋膜源性细胞获得成纤维细胞样外观(图3)。事实上，当结蛋白(肌原细胞标志物)和波形蛋白(成纤维细胞标志物)染色时，几乎所有的筋膜源性细胞均表达波形蛋白，而几乎不表达结蛋白、CD34，v-WF(内皮标记)和α平滑肌肌动蛋白(周细胞标记)，支持这些细胞的成纤维细胞的特性[18-19]。 2.3 筋膜源性细胞与大鼠L6成肌细胞混合培养分析不同比例的筋膜源性细胞和L6成肌细胞混合群在软骨培养检测结果表明，其成软骨能力随着L6成肌细胞比例的增加而逐渐下降。完全由筋膜源性细胞组成的细胞团表现出最强的成软骨能力，其软骨细胞形态，及细胞外基质形成方面符合体外培养软骨细胞的形态学改变，而完全由L6成肌细胞组成的细胞团表现出最弱的软骨形成潜力，其软骨细胞形态，大小及细胞基质形成方面较差(图4)。在较高放大倍率下，理想的软骨形成分化(筋膜源性细胞)和低分化(L6)的比较，在番红O阳性染色(红色)中，纯筋膜源性细胞组可以见到典型的软骨基质内占据空隙的软骨细胞，而在纯L6组中没有发现典型的软骨细胞。番红O和阿尔新蓝染色后表明其筋膜源性细胞是主要的成软骨细胞，而L6成肌细胞表现出的成软骨能力十分有限。"

References

[1] Borakati A, Mafi R, Mafi P, et al. A systematic review and meta-analysis of clinical trials of mesenchymal stem cell therapy for cartilage repair. Curr Stem Cell Res Ther. 2018; 13(3):215-225.

[2] Cucchiarini M, Thurn T, Weimer A, et al. Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9. Arthritis Rheum.2007;56(1):158-167.

[3] Jo CH, Lee YG, Shin WH, et al. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Cells. 2014; 32(5):1254-1266.

[4] Van Manen MD, Nace J, Mont MA. Management of primary knee osteoarthritis and indications for total knee arthroplasty for general practitioners. J Am Osteopath Assoc. 2012; 112(11):709-715.

[5] Cucchiarini M, Madry H. The potential of gene transfer for the treatment of osteoarthritis. Regen Med. 2014;9(1): 5-8.

[6] Park BW, Kang EJ, Byun JH, et al. In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues. Differentiation. 2012; 83(5): p. 249-59.

[7] Wakitani S, Imoto K, Yamamoto T, et al. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage. 2002;10(3):199-206.

[8] Chew E, Prakash R, Khan W. Mesenchymal stem cells in human meniscal regeneration: A systematic review. Ann Med Surg (Lond). 2017; 24:3-7.

[9] Kaul G, Cucchiarini M, Arntzen D, et al. Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo. J Gene Med. 2006; 8(1):100-111.

[10] Chamberlain D, Fox GJ, Ashton B, et al. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells.2007; 25(11):2739-2749.

[11] Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005;52(8): 2521-2529.

[12] Li G, Corsi-Payne K, Zheng B, et al. The dose of growth factors influences the synergistic effect of vascular endothelial growth factor on bone morphogenetic protein 4-induced ectopic bone formation. Tissue Eng Part A. 2009;15(8): 2123-2133.

[13] Mamidi MK, Das AK, Zakaria Z, et al. Mesenchymal stromal cells for cartilage repair in osteoarthritis. Osteoarthritis Cartilage. 2016; 24(8):1307-16.

[14] Kuroda R, Usas A, Kubo S, et al. Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells. Arthritis Rheum. 2006; 54(2):433-442.

[15] Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 2008; 3(9): 1501-1509.

[16] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest. 2002; 110(6): 751-759.

[17] Kristjansson B, Honsawek S. Mesenchymal stem cells for cartilage regeneration in osteoarthritis. World J Orthop. 2017; 8(9): 674-680.

[18] Neuhuber B, Swanger SA, Howard L, et al., Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol. 2008;36(9): 1176-1185.

[19] Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008; 3(3): 301-313.

[20] Li G, Zheng B, Meszaros LB, et al., Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle. J Mol Cell Biol. 2011;3(6): 369-377.

[21] Davatchi F, Abdollahi BS, Mohyeddin M, et al., Mesenchymal stem cell therapy for knee osteoarthritis. Preliminary report of four patients. Int J Rheum Dis. 2011; 14(2): 211-215.

[22] Crema MD, Roemer FW, Marra MD, et al. Articular cartilage in the knee: current MR imaging techniques and applications in clinical practice and research. Radiographics. 2011; 31(1): 37-61.

[23] Battaglia M, Rimondi E, Monti C, et al. Validity of T2 mapping in characterization of the regeneration tissue by bone marrow derived cell transplantation in osteochondral lesions of the ankle. Eur J Radiol. 2011;80(2): e132-139.

[24] Shimomura K, Ando W, Tateishi K, et al. The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model. Biomaterials. 2010;31(31): 8004-8011.

[25] Wang Y, Yu D, Liu Z, et al. Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix. Stem Cell Res Ther. 2017;8(1):189.

[26] Yu HC, Peng H, He XS, et al. Comparison of short- and long-term outcomes after extralevator abdominoperineal excision and standard abdominoperineal excision for rectal cancer: a systematic review and meta-analysis. Int J Colorectal Dis.2014;29(2):183-191.

[27] Noth U, Steinert AF, Tuan RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Clin Pract Rheumatol. 2008;4(7):371-380.

[28] Kragh JF, Svoboda SJ, Wenke JC, et al. Epimysium and perimysium in suturing in skeletal muscle lacerations. J Trauma. 2005;59(1):209-212.

[29] Kurose T, Asai Y, Mori E, et al. Distribution and change of collagen types I and III and elastin in developing leg muscle in rat. Hiroshima J Med Sci. 2006; 55(3): 85-91.

[30] Grande DA, Pitman MI, Peterson L, et al. The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J Orthop Res. 1989;7(2): 208-218.

[31] Evans CH, Ghivizzani SC, Robbins PD. Arthritis gene therapy and its tortuous path into the clinic. Transl Res. 2013;161(4): 205-216.