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MESENCHYMAL STEM CELLS FOR TISSUE REGENERATION
Guang Yang, Song Li and Ngan F. Huang
1.Introduction
In the past two decades, significant progress has been made in the field of stem cell research. An important finding is that adult stem cells harbor greater regeneration potential and plasticity than what was previously thought. This discovery has led to tremendous research interest in developing methods to direct stem cell differentiation into lineages for the therapeutic delivery into diseased or dysfunctional tissues. Among the adult stem cells, mesenchymal stem cells (MSCs) are a promising therapeutic cell source due to the ease of isolation, high proliferative capacity, and multipotency.1 MSCs can be found in numerous tissues of the adult mammal and can be harvested in large quantities by minimally invasive and reproducible approaches. Therapeutic MSCs can be delivered directly in vivo or incorporated into tissue engineered constructs in vitro before transplantation. Both of these two approaches have been explored for treating a wide range of diseases or traumatic events, including myocardial infarction, peripheral arterial disease, spinal cord injury, musculoskeletal system defects, and skin wounds. By developing robust methods of differentiating MSCs into therapeutic cells of interest and organizing the cells into functional three-dimensional tissues, it may be possible to fulfill the potential of MSCs for clinical use. This review aims to provide an overview of some therapeutic applications for MSCs in tissue engineering and regenerative medicine.
2.MSC Sources and Phenotype
MSCs can be generally defined as adherent and elongated cells that reside in mesenchymal tissues and can self-renew as well as produce progeny with more specialized functions. MSCs have been observed and purified from numerous origins, including bone marrow, adipose tissue, skeletal muscle, blood, liver spleen and dental pulp.2–8 This review primarily focuses on MSCs derived from bone marrow and adipose tissues, as they are the most well characterized origins of MSCs among all known MSC sources.
2.1Bone marrow MSCs
Although MSCs account for only 0.01% among total nucleated cells in the bone marrow, they have over a million-fold expansion capability and multilineage differentiation potential.9 Bone marrow MSCs are easily harvested by aspiration from the iliac crest. Phenotypically, there is no unique single marker that specifically identifies bone marrow MSCs. Consequently, MSCs are characterized based on the positive expression of numerous cell surface antigens such as CD29 (integrin β1), CD44 (receptor for hyaluronic acid and matrix proteins), CD 105 (endoglin), and CD166 (cell adhesion molecule).10 On the other hand, they do not express markers typically associated with hematopoietic cells, such as CD14 (monocyte surface antigen), CD34 (hematopoietic stem cell surface antigen), and CD45 (leukocyte surface antigen).1 Due to the differences in characterization methods, the International Society for Cellular Therapy suggested the recommended designation of these cells as multipotent mesenchymal stromal cells and proposed the following minimal criteria for MSCs: adherence to plastic dishes; phenotypic expression of CD105, CD73, and CD90; lack of surface molecule expression of CD45, CD34, CD14, or CD11b; CD79α or CD19 and class II major histocompatibility complex antigen (HLA-DR); and differentiation capacity into osteoblasts, adipocytes, and chondroblasts in vitro.11
Based on the cell surface antigens, bone marrow MSCs can be isolated using fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). Other methods employed to purify MSCs include Percoll gradient centrifugation and selective adherence onto tissue-culture treated Petri dishes. To maintain their proliferative capacity, the purified bone marrow MSCs can be expanded in culture media containing defined serum-free components (i.e. StemPro® MSC Serum Free Medium, Invitrogen, Carlsbad, CA) or pre-screened fetal bovine serum. Under these growth conditions, bone marrow MSCs can be cultured for more than five passages with negligible changes in phenotype. However, the differentiation potential of bone marrow MSCs seems passage-dependent: loss of osteogenic and adipogenic potential was found along the passaging of cells.12,13
2.2Adipose-derived stem cells
According to the definition established by the International Fat Applied Technology Society, adipose-derived stem cells (ASCs) are the plastic-adherent, multipotent cell population isolated from the stromal vascular fraction (SVF) of adipose tissue.14 These cells were firstly found capable of differentiating into adipogenic, chondrogenic, and osteogenic lineages,15 and were later confirmed to give rise to other cell types, including endothelial cells (ECs), smooth muscle cells (SMCs) and cardiomyocytes.16–18 To isolate ASCs, adipose tissue derived from liposuction is digested with collagenase and then centrifuged to separate the SVF pellet from the adipocytes in the upper layer.15 When cultured under standardized conditions, SVF is homogenized, giving rise to the pure population of ASCs.19 Like bone marrow MSCs, ASCs can be purified based on the expression profile of surface marker antigens using FACS or MACS. ASCs can be maintained in defined serum-free medium (i.e. MesenPRO RS Media, Invitrogen, CA) as well as serum-containing media.
Although ASCs and bone marrow MSCs present more than 90% similarity in immunophenotype,20 there are some reported differences in surface antigen expression. For example, CD14 and HLA-DR were reported to be absent in bone marrow MSCs, but have been identified in early-passage human ASCs at low frequency.21 Furthermore, ASCs appear to have temporal changes in immunophenotype with subsequent passaging.21 However, these differences in immunophenotype could also be attributed to differences in species or the methods of isolation, purification, or detection.
3.Differentiation of MSCs in vitro
The application of MSCs to tissue engineering and regenerative medicine often involves the pre-differentiation of cells in vitro into lineages of interest before delivering them in vivo for therapeutic treatment. Bone marrow MSCs and ASCs have been shown to differentiate into a variety of lineages, including myogenic, osteogenic, chondrogenic, and adipogenic lineages.22 A number of strategies to direct their differentiation have been used, including the use of soluble factors, mechanical stimulation, extracellular matrix (ECM) factors, and genetic engineering approaches, which are briefly discussed below.
One of the most commonly used strategies to induce differentiation is to using soluble factors such as growth factors and small molecules. Using soluble factors, bone marrow MSCs and ASCs have a high propensity to differentiate into cells of mesenchymal lineage, including bone, adipose tissue, and cartilage. Osteogenic differentiation can be induced by culturing the cells in the presence of dexamethasone, ascorbic acid, and β-glycerophosphate.1 Adipogenesis is usually accomplished by treatment with DMEM supplemented with FBS, dexamethasone, 3-isobutyl-1-methylxanthine, and 1x insulin-transferrin-selenium (ITS).23 Chondro-genesis of high-density MSC pellet cultures is induced using serum-free DMEM supplemented with dexamethasone, L-proline, ascorbate, and transforming growth factor β1 or 3 (TGF-β1/3). In addition, bone morphogenetic protein 6 (BMP-6) is added to the chondrogenic medium to improve the chondrogenesis of ASC by rescuing the expression of TGF-β1.24 Besides osteogenic, adipogenic, and chondrogenic lineages, MSCs have been shown to differentiate toward other lineages at lower yields. For example, platelet-derived growth factor (PDGF) and TGF-β3 stimulate smooth muscle phenotype,25 whereas 5-azacytidine treatment induces the formation of cardiac-like cells that expresses cardiac markers β-myosin heavy chain, desmin, and α-cardiac actin.26
Besides soluble factors, mechanical stimulation is another potent regulator of cell behavior and function. Physiologically, mechanical s...
