In vitro osteogenesis from human skin-derived precursor cells

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.     

Myelin repair: the role of stem and precursor cells in multiple sclerosis

Multiple sclerosis is the most common potential cause of neurological disability in young adults. The disease has two distinct clinical phases, each reflecting a dominant role for separate pathological processes: inflammation drives activity during the relapsing-remitting stage and axon degeneration represents the principal substrate of progressive disability. Recent advances in disease-modifying treatments target only the inflammatory process. They are ineffective in the progressive stage, leaving the science of disease progression unsolved. Here, the requirement is for strategies that promote remyelination and prevent axonal loss. Pathological and experimental studies suggest that these processes are tightly linked, and that remyelination or myelin repair will both restore structure and protect axons. This review considers the basic and clinical biology of remyelination and the potential contribution of stem and precursor cells to enhance and supplement spontaneous remyelination.     

Polypeptide factors regulating osteogenesis and bone marrow repair

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation. © 1994 Wiley-Liss, Inc.     

Osteogenic stem cells of the bone marrow

This paper presents literature and author's own data demonstrating that bone marrow contains determined osteogenic precursor cells with high potential to differentiation. They are stem cells of the bone and belong to the stromal cell line of the bone marrow which is histogenetically independent of hemopoietic cells. The paper presents detailed analysis of bone marrow stromal cells (CFUf) as well as of their osteogenic properties and requirements in growth factors. In conclusion mutual growth-stimulating interactions in the system of hemopoietic stromal cells are reviewed.     

Osteogenic properties of human myogenic progenitor cells

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.     

Osteogenic potential of rat spleen stromal cells

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.     

Compactin enhances osteogenesis in murine embryonic stem cells

Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and beta-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation.     

Osteogenic differentiation of human dental papilla mesenchymal cells

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.     

Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold

This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.     

Reproduction and differentiation of the cells in enchondral osteogenesis

Light and electron-microscopical 3H-thymidine autoradiography was used to study the dynamics of cell populations in the zones of enchondral osteogenesis in a tubular bone. In the early postnatal ontogenesis little differentiated perivascular cells are characterized by the highest proliferative activity in this region; they are considered as a population containing initial forms of the histogenetic sequence (differon) of the stromal fibroblast-like cells including osteoblasts. Differentiation of osteogenic cells from the initial forms to the mature osteoblasts proceeds through a number of successive divisions (1-3 divisions) and is accompanied by a decrease in the proliferative activity due to the increase in the generation time and decrease in the cell proliferative pool. The major part of osteoblasts is outside the mitotic cycle. At the later stages of ontogenesis the intensity of growth processes in the bone is provided for by changes in the proliferative pool of the committed precursor cells (preosteoblasts) which make a part of endosteum, vascular channels and bone marrow stroma.     

Osteogenic comparison of expanded and uncultured adipose stromal cells

Background aimsAdipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC.MethodsAdipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard protocols or prepared by a commercial vendor using proprietary technology (proprietary stromal vascular fraction, SVFp). Cells were seeded on collagen sponges and maintained in osteogenic culture conditions for up to 21 days to assess osteogenic potential. The ability of each population to stimulate neovascularization and bone healing was determined upon implanting cell-loaded sponges into a rodent calvarial bone defect. Neovascularization was measured 3 weeks post-implantation, while bone formation was monitored over 12 weeks using in vivo microcomputed tomography (microCT).ResultsSVFp exhibited increased intracellular alkaline phosphatase activity compared with cultured ASC but proliferated minimally. Histologic analysis of explanted tissues demonstrated greater vascularization in defects treated with cultured ASC compared with SVFp. We detected increases in bone volume for defects treated with cultured cells while observing similar values for bone mineral density, regardless of cell type.ConclusionsThese results suggest that expanded ASC are advantageous for neovascularization and bone healing in this model compared with SVFp, and provide additional evidence of the utility of ASC in bone repair.     

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.     

Progenitor cells in vascular repair

PURPOSE OF REVIEW: A common characteristic of all types of vascular disease is endothelial dysfunction/damage followed by an inflammatory response. Although mature endothelial cells can proliferate and replace damaged cells in the vessel wall, recent findings indicate an impact of stem and progenitor cells in repair process. This review aims to briefly summarize the recent findings in stem/progenitor cell research relating to vascular diseases, focusing on the role of stem/progenitor cells in vascular repair. RECENT FINDINGS: It has been demonstrated that endothelial progenitor cells present in the blood have an ability to repair damaged arterial-wall endothelium. These cells may be derived from a variety of sources, including bone marrow, spleen, liver, fat tissues and the adventitia of the arterial wall. In response to cytokine released from damaged vessel wall and adhered platelets, circulating progenitor cells home in on the damaged areas. It was also reported that the adhered progenitor cells can engraft into endothelium and may differentiate into mature endothelial cells. SUMMARY: Vascular progenitor cells derived from different tissues have an ability to repair damaged vessel, in which the local microenvironment of the progenitors plays a crucial role in orchestrating cell homing and differentiation.