Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

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Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT‐PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage‐specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and western blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.

Functional engineering of musculoskeletal tissues generally involves rapid expansion of progenitor cells in vitro while retaining their potential for further differentiation and then induction in specific culture conditions. The autologous adipose-derived stromal cells (ASCs) are considered to contain pluripotent mesenchymal stem cells. Imaging with expression of green fluorescent protein (GFP) facilitates the detailed research on ASCs physiological behavior during differentiation into a variety of cell lineages both in vitro and in vivo. In this study, we aimed to confirm the trans-germ plasticity of homogeneously marked ASCs from GFP transgenic mice. Simultaneously, the term and intensity of GFP expression in ASCs were also focused on during variant inductions, when cells were incubated with multiple growth factors and adjuvant. ASCs were harvested from inguinal fat pads of transgenic nude mice, passaged 3 times in monolayer cultures, and then transferred to osteogenic, adipogenic, neurogenic, and myogenic medium. The morphological characterization of inductive cells was observed using phase-contrast microscopy and histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The expression of marker genes or proteins was measured using RT-PCR and immunocytochemical analysis. Collagen type I, osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, peroxisome proliferator-activated receptor(PPAR)-gamma2 and lipoprotein lipase (LPL) were positive in adipogenic ones, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) were positive in neurogenic ones, and alpha-smooth muscle actin (alpha-SMA) was positive in myogenic ones. Moreover, the results of fluorescence microscopic imaging suggested that there was no significant decline of GFP expression during ASCs differentiation and the level of GFP maintained stable till differentiated ASCs showed apoptotic phenotype. So the endogenous GFP and multilineage potential of transgenic ASCs had no influences on each other. Since the population of GFP ASCs can be easily identified, it is proposed that they may be promising candidate seed cells for further studies on ASCs tissue engineering, especially the study on engineered tissues formed in vivo.