Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.     

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.     

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments.     

PDZRN3 Negatively Regulates BMP-2�Cinduced Osteoblast Differentiation through Inhibition of Wnt Signaling

PDZRN3 is a member of the PDZ domain–containing RING finger family of proteins. We previously showed that PDZRN3 is essential for the differentiation of C2C12 mouse mesenchymal progenitor cells into myotubes. Mesenchymal progenitor cells differentiate into osteoblasts, chondrocytes, and adipocytes in addition to myotubes, and we have now examined the potential role of PDZRN3 in the differentiation of C2C12 cells into osteoblasts. The abundance of PDZRN3 in C2C12 cells was increased after the induction of osteoblast differentiation by exposure to bone morphogenetic protein (BMP)-2 in low-serum medium. Depletion of PDZRN3 in C2C12 cells by RNA interference resulted in marked enhancement of the BMP-2–induced up-regulation of alkaline phosphatase (ALP) activity. Dkk1, an inhibitor of Wnt signaling, markedly attenuated the enhancement of the BMP-2–induced increase in ALP activity by PDZRN3 depletion. The up-regulation of ALP activity by Wnta3a was also promoted by depletion of PDZRN3. Furthermore, the expression and Wnt3a-induced phosphorylation of LRP6 as well as the increase in the cytosolic abundance of β-catenin induced by Wnt3a were potentiated in PDZRN3-depleted cells. These results indicate that PDZRN3 plays an important role in negative feedback control of BMP-2–induced osteoblast differentiation in C2C12 cells through inhibition of Wnt–β-catenin signaling.     

具有间充质干细胞特征的CD105+细胞在胎儿多种组织器官的分布

CD105是骨髓间充质干细胞的特征性表型之一。为了研究机体各组织器官也存留有间充质干细胞,首先检测胎儿各组织CD105^ 细胞的分布,进而分离胎儿各组织CD105^ 细胞。将CD105^ 细胞向脂肪和成骨细胞诱导分化。结果表明胎儿心、肝、肺、血管、肌肉、皮肤等组织含有CD105^ 间充质干细胞。在间充质干细胞分化为脂肪细胞时,CD105表达明显下降。地塞米松可以促进脂肪细胞形成并提高了培养液中甘油三酯的含量。而向成骨细胞分化时,诱导的成骨细胞胞浆内外有电子密度高的钙盐沉积。以上结果提示,分布于多种组织的间充质干细胞异常分化可能与疾病的发生有关。     

Osteoblastic cells: differentiation and trans-differentiation

The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.     

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

Isolation and characterization of mesenchymal stem cells from chicken bone marrow

The bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells, which can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit and other mammals, but rarely in poultry. So, in this study, Thirty- to sixty-day old chicken was chosen as experimental animal, to isolate and characterize BMSCs from them. To investigate the biological characteristics of chicken BMSCs, immunofluorescence and RT-PCR were used to detect the characteristic surface markers of BMSCs. Growth curves were drawn in accordance with cell numbers. To assess the differentiation capacity of the BMSCs, cells were induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The surface markers of BMSCs, CD29, CD44, CD31, CD34, CD71 and CD73, were detected by immunofluorescence and RT-PCR assays. The growth curves of different passages were all typically sigmoidal. Karyotype analysis showed that these in vitro cultured cells were genetically stable. In addition, BMSCs were successfully induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The results suggest that the BMSCs isolated from chicken possess similar biological characteristics with those separated from other species, and their multi-lineage differentiation potentiality herald a probable application for cellular transplant therapy in tissue engineering.