Search

Fetal bovine serum (FBS) is widely used in cell cultures due to its high stability and easy access. It was also used as a substitute for porcine follicular fluid (PFF) in previous studies. However, FBS components are unclear, and the presence of FBS in culture media may introduce a variation from batch to batch. This study aimed to establish an effective method to screen FBS in place of PFF in the culture media for porcine oocytes in vitro. We screened FBS from different sources by using porcine fetal fibroblast cells. The effects of six FBS samples on porcine fetal fibroblast cell growth were tested via frozen cell survival assay, cell clone formation assay, cell growth curve, and cell passage activity assay. The best serum that we called GFBS (heat‐inactivated FBS, cat. no. 10500‐64; Gibco) showed a similar effect on the maturation and development of porcine oocytes to that of PFF and can be used as a good substitute for PFF. These results suggested that the porcine fetal fibroblast cell culture test can be used as a valuable method to screen FBS for porcine oocyte maturation and embryonic development in vitro.

Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+Lepr +Sca-1+CD146+ and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo.

The objective of this study is to assess water environment and ecological risks from heavy metals of soils in Hanjiang River Basin, China. We carried out investigations on the changing patterns of spatiotemporal distribution of soil chemical elements, and further identified the changing behaviors, geochemical process and driving forces of heavy metals in soil by integrated analysis of natural environmental factors and human activities. In order to achieve these goals, we used field investigation, laboratory testing and GIS spatial simulation. The following findings were achieved. Firstly, general soil weathering in the Hanjiang River Basin basically evolves from Ca and Na stages to K stage. Ca and Na decreased rapidly, and silicate weathering evolved gradually. Major ions are subject to carbonate dissolution. About 2/3 of the soils in the basin are alkaline (pH > 7) which provide good conditions for mineralization of organic nitrogen. Secondly, specific soil weathering processes differ in spatial patterns and element transport, driven by integrated interior forces (e.g., mineral materials) and external forces (e.g., temperature, precipitation, wind, and gravity). Original mineral materials are changed into soils with different physical properties (soil texture, mechanical composition) and chemical components (organic matter). Upstream original mineral materials have been transported, accumulated, and deposited downstream, driven by forces of gravity, water transport, glacial effects, and wind. Thirdly, key ecological factors of plant growth change greatly. Increase of soil humus mitigates pollution hazards and enhances soil self-purification. Land use and cover is one of the key contributors to determine geochemical patterns of soil organic matter. Intensity of mineral weathering decreases with the decrease of biomass. The comprehensive pollution index and ecological risk index increased from northwest to southeast, corresponding to increased erosive rainfall and rainstorms more than human activities.

Utilization of small molecules in modulation of stem cell self-renewal is a promising approach to expand stem cells for regenerative therapy. Here, we identify Icaritin, a phytoestrogen molecule enhances self-renewal of mouse embryonic stem cells (mESCs). Icaritin increases mESCs proliferation while maintains their self-renewal capacity in vitro and pluripotency in vivo. This coincides with upregulation of key pluripotency transcription factors OCT4, NANOG, KLF4 and SOX2. The enhancement of mESCs self-renewal is characterized by increased population in S-phase of cell cycle, elevation of Cylin E and Cyclin-dependent kinase 2 (CDK2) and downregulation of p21, p27 and p57. PCR array screening reveals that caudal-related homeobox 2 (Cdx2) and Rbl2/p130 are remarkably suppressed in mESCs treated with Icaritin. siRNA knockdown of Cdx2 or Rbl2/p130 upregulates the expression of Cyclin E, OCT4 and SOX2, and subsequently increases cell proliferation and colony forming efficiency of mESCs. We then demonstrate that Icaritin co-localizes with estrogen receptor alpha (ERα) and activates its nuclear translocation in mESCs. The promotive effect of Icaritin on cell cycle and pluripotency regulators are eliminated by siRNA knockdown of ERα in mESCs. The results suggest that Icaritin enhances mESCs self-renewal by regulating cell cycle machinery and core pluripotency transcription factors mediated by ERα.

Peripheral blood-derived multipotent mesenchymal stromal cells circulate in low number. They share, most although not all, of the surface markers with bone marrow-derived multipotent mesenchymal stromal cells, possess diverse and complicated gene expression characteristics, and are capable of differentiating along and even beyond mesenchymal lineages. Although their origin and physio-pathological function are still unclear, their presence in the adult peripheral blood might relate to some interesting but controversial subjects in the field of adult stem cell biology, such as systemic migration of bone marrow-derived multipotent mesenchymal stromal cells and the existence of common hematopoietic-mesenchymal precursors. In this review, current studies/knowledge about peripheral blood-derived multipotent mesenchymal stromal cells is summarized, and the above-mentioned topics are discussed.

Mesenchymal stem cells (MSCs) were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. Whether or not the PB derived MSCs (PBMSCs) could enhance the regeneration of large bone defects have not been reported. In this study, rabbit MSCs were obtained from mononuclear cells (MNCs) cultures of both the PB and bone marrow (BM) origin. The number of PBMSCs was relatively lower, with the colony forming efficiency (CFE) ranging from 1.2 to 13 per million MNCs. Under specific inductive conditions, PBMSCs differentiated into osteoblasts, chondrocytes, and adipocytes, showing multidifferentiation ability similar to BMMSCs. Bilateral 20 mm critical-sized bone defects were created in the ulnae of 12 6-month-old New Zealand white rabbits. The defects were treated with allogenic PBMSCs/Skelite (porous calcium phosphate resorbable substitute), BMMSCs/Skelite, PBMNCs/Skelite, Skelite alone, and left empty for 12 weeks. Bone regeneration was evaluated by serial radiography, peripheral quantitative computed tomography (pQCT), and histological examinations. The X-ray scores and the pQCT total bone mineral density in the PBMSCs/Skelite and BMMSCs/Skelite treated groups were significantly greater than those of the PBMNCs/Skelite and Skelite alone groups ( p

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor (IR) selectively in chondrocytes (CartIR (-/-)) had no discernable differences in total femoral length compared to control littermates. However, CartIR (-/-) mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor (IGF)-1R mRNA and protein levels. Subsequently, IGF-1 induced phosphorylation of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR (-/-) mice when compared with that of the control littermates, which was associated with upregulation of tuberous sclerosis complex 2 (TSC2). These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks post-transplantation shows that 3D complexes incorporated with Icariin significantly enhance articular cartilage repair with higher scores particularly in selected parameters including SO+ cartilage area, subchondral bone and overall assessment than that of the controls. The results suggest that Icariin may inhibit PHD activity likely through competition for cellular iron ions and therefore it may serve as an HIF-1α activator to promote articular cartilage repair through regulating chondrocyte proliferation, differentiation and integration with subchondral bone formation.

To obtain enough quantity of osteogenic cells is a challenge for successful cell therapy in bone defect treatment, and cell numbers were usually achieved by culturing bone marrow cells in a relatively long duration. This study reports a simple and cost-effective method to enhance the number of mesenchymal stem cells (MSCs) by collecting and replating the nonadherent cell population of marrow MSCs culture. Bone marrow MSCs were isolated from 11 patients, cultured at a density of 1 x 10(5)/cm(2) to 1 x 10(6)/cm(2) in flasks. For the first three times of media change, the floating cells were centrifuged and replated in separate flasks. The total number of cells in both the primary and replating flasks were counted at day 21. Cell proliferation rate, potentials for osteogenic, chondrognenic, and adipogenic differentiation were examined in both cell types in vitro. In vivo osteogenic potentials of the cells were also tested in mice implantation model. The results showed that MSCs derived from nonadherent cell population of marrow cell cultures have similar cell proliferation and differentiation potentials as the originally attached MSCs in vitro. When implanted with hydroxyapatite/tricalcium phosphate (HA-TCP) materials subcutaneously in serve combined immune deficiency (SCID) mice, newly formed bony tissues were found in both cell type groups with osteocalcin expression. We have obtained 36.6% (20.70%-44.97%) more MSCs in the same culture period when the nonadherent cell populations were collected. The findings confirmed that the nonadherent cell population in the bone marrow culture is a complementary source of MSCs, collecting these cells is a simple and cost-effective way to increase MSCs numbers and reduce the time required for culturing MSCs for clinical applications.

During bone development and repair, angiogenesis, osteogenesis, and bone remodeling are closely associated processes that share some common mediators. In the present study nonadherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP-positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, and 300 ng/mL) and rhVEGF (0 and 25 ng/mL) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential, and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/mL showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/mL) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/mL rhBMP-2. However, rhVEGF-induced resorptive function was inhibited by 30 ng/mL and 300 ng/mL rhBMP-2 in a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis; exact mechanisms are yet to be elucidated. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.