Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells.

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic ß-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.

[Down-Turner syndrome (45,X/47,XY,+21): case report and review].

We report the case of a 3-yr-old boy with Down-Turner mosaicism and review the previous reports of Down-Turner syndrome with documented karyotyping and clinical features. The patient showed clinical features of Down syndrome without significant stigma of Turner syndrome. Cytogenetic analysis of peripheral blood preparations by using G-banding revealed mosaicism with 2 cell lines (45,X[29]/47,XY,+21[4]). FISH analysis revealed that 87.5% of the cells had monosomy X karyotype and 12.5% of the cells had XY karyotype; trisomy 21 was only detected in the Y-positive cells. We suggest that additional cells should be analyzed and molecular genetic studies should be conducted to rule out double aneuploidy when karyotypes with sex chromosome aneuploidies and mosaicism are encountered, as in our case of Down syndrome mosaic with sex chromosome aneuploidy.

Selection of optimal passage of bone marrow-derived mesenchymal stem cells for stem cell therapy in patients with amyotrophic lateral sclerosis.

Mesenchymal stem cells (MSCs) obtained from bone marrow (BM) are currently used as an alternative therapy in amyotrophic lateral sclerosis (ALS) patients. Selection of optimal passages of autologous BM-derived MSCs during long-term in vitro expansion is important for clinical trials in patients with ALS. We isolated and expanded MSCs from the BM of eight ALS patients to analyze the growth kinetics, differentiation potential, cellular surface antigen expression, karyotype modifications and secretion of various cytokines during long-term culture. The morphology and size of the cells changed from small and spindle-like cells to large and polygonal types in later passages. The growth rate of the MSCs was highest in the third passage, followed by a gradual decrease. There were no special modifications of cell surface antigens or the karyotype of the MSCs from the first to the tenth passage. MSCs in the fourth passage were differentiated into adipocytes, osteocytes and chondrocytes. When we analyzed the cultured media of MSCs at the third, fifth, seventh and ninth passages, IL-6, VEGF and IL-8 showed high expression, with more than 50pg/10,000 cells at these passages; however, their expression progressively decreased with additional passages. In addition, secretion of IL-15, GM-CSF, IL-10, PDGF-bb, G-CSF, IL-1beta, basic FGF and IFN-gamma gradually decreased over prolonged culture. We suggest that MSCs at earlier passages are more suitable for stem cell therapy in ALS patients because of their stability and more potent anti-inflammatory and neuroprotective properties.

Secretory profiles and wound healing effects of human amniotic fluid-derived mesenchymal stem cells.

Recent evidence shows that amniotic fluid (AF) contains multiple cell types derived from the developing fetus, and may represent a novel source of stem cells for cell therapy. In this study, we examined the paracrine factors released by human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) and their ability to accelerate the wound-healing process by stimulating proliferation and migration of dermal fibroblasts. AF-MSCs expressed the typical MSC marker proteins CD13, CD29, and CD44 and differentiated into adipocytes, osteoblasts, and chondrocytes when exposed to the appropriate differentiation media. In addition, AF-MSC-conditioned media (AF-MSC-CM) significantly enhanced proliferation of dermal fibroblasts. Antibody-based protein array and enzyme-linked immunosorbent assay (ELISA) indicated that AF-MSC-CM contains various cytokines and chemokines that are known to be important in normal wound healing, including IL-8, IL-6, TGF-beta, TNFRI, VEGF, and EGF. Application of AF-MSC-CM significantly enhanced wound healing by dermal fibroblasts via the TGF-beta/SMAD2 pathway. Levels of p-SMAD2 were increased by AF-MSC-CM, and both the increase in p-SMAD2 and migration of dermal fibroblasts were blocked by inhibiting the TGF-beta/SMAD2 pathway. Moreover, in a mouse excisional wound model, AF-MSC-CM accelerated wound healing. These data provide the first evidence of the potential for AF-MSC-CM in the treatment of skin wounds.