Fibrillin‐1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age‐related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin‐1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin‐1 (Fbn1^Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1^Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1^Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1‐silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin‐1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1^Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin‐1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research.     

Adverse Fat Depots and Marrow Adiposity Are Associated With Skeletal Deficits and Insulin Resistance in Long‐Term Survivors of Pediatric Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem‐cell transplantation (alloHSCT) survivors treated with total body irradiation (TBI) exhibit bone deficits and excess adiposity, potentially related to altered mesenchymal stem cell differentiation into osteoblasts or adipocytes. We examined associations among fat distribution, bone microarchitecture, and insulin resistance in alloHSCT survivors after TBI. This was a cross‐sectional observational study of 25 alloHSCT survivors (aged 12 to 25 years) a median of 9.7 (4.3 to 19.3) years after alloHSCT compared to 25 age‐, race‐, and sex‐matched healthy controls. Vertebral MR spectroscopic imaging and tibia micro‐MRI were used to quantify marrow adipose tissue (MAT) and trabecular microarchitecture. Additional measures included DXA whole‐body fat mass (WB‐FM), leg lean mass (Leg‐LM), trunk visceral adipose tissue (VAT), and CT calf muscle density. Insulin resistance in alloHSCT survivors was estimated by HOMA‐IR. AlloHSCT survivors had lower Leg‐LM (p < 0.001) and greater VAT (p < 0.01), MAT (p < 0.001), and fat infiltration of muscle (p = 0.04) independent of WB‐FM, versus matched controls; BMI did not differ. Survivors had lower bone volume fraction and abnormal microarchitecture including greater erosion and more rod‐like structure versus controls (all p = 0.04); 14 had vertebral deformities and two had compression fractures. Greater WB‐FM, VAT, MAT, and muscle fat infiltration were associated with abnormal trabecular microarchitecture (p < 0.04 for all). AlloHSCT HOMA‐IR was elevated, associated with younger age at transplantation (p < 0.01), and positively correlated with WB‐FM and VAT (both p < 0.01). In conclusion, the markedly increased marrow adiposity, abnormal bone microarchitecture, and abnormal fat distribution highlight the risks of long‐term treatment‐related morbidity and mortality in alloHSCT recipients after TBI. Trabecular deterioration was associated with marrow and visceral adiposity. Furthermore, long‐term survivors demonstrated sarcopenic obesity, insulin resistance, and vertebral deformities. Future studies are needed to identify strategies to prevent and treat metabolic and skeletal complications in this growing population of childhood alloHSCT survivors. © 2015 American Society for Bone and Mineral Research.     

085Bone Marrow Mesenchymal Stem Cells as a Potential Source to Repair the Injury Skin

Adult stem cells are found in various tissues and organs and have potential to differentiate into different cell lineages, including bone, cartilage, fat, tendon, muscle, epithelial cells of the skin and gastrointestinal tract, etc. Here, we report that the expended and purified bone marrow MSCs might take on phenotypes and characteristics of vascular endothelial cells or epidermal cells after cultured and induced with different lineage‐specific culture conditions in vitro. Also, in vivo grafting experiments confirmed that those labeled MSCs could get the phenotypes of vascular endothelial cells in granulation tissue and sebaceous duct cells and epidermal cells in regenerated skin, which imply that these grafted MSCs might have transdifferentiated into vascular endothelial cells and sebaceous duct cells and epidermal cells. Our results indicate that locally delivered bone marrow MSCs may generate de novo intact skin and get perfect skin regeneration after full thickness injury.     

骨髓基质干细胞成骨-成脂分化及补肾中药对其影响的研究进展

骨髓基质干细胞在不同条件的诱导下，可以分化为成骨细胞、软骨细胞、成纤维细胞、脂肪细胞、神经细胞等多种细胞系，在骨髓微环境中骨和脂肪形成之间的关系是复杂的，成骨细胞与脂肪细胞均来源于骨髓基质干细胞 (MSCs)，并且在其向成骨细胞和脂肪细胞分化之间存在相互逆转的关系和很大程度的可塑性。骨质疏松患者骨量减少与骨髓腔中脂肪组织增加有关，这可能与骨髓中MSCs分化失衡，过多向脂肪细胞分化有关。中医从“肾主骨”、“髓生骨”理论出发，认为肾精不足所致的骨髓空虚是骨质疏松症发病的关键，补肾中药通过影响骨髓基质干细胞成骨-成脂分化，从而防止骨质疏松症的发生。     

Effect of Low‐Magnitude Mechanical Stimuli on Bone Density and Structure in Pediatric Crohn's Disease: A Randomized Placebo‐Controlled Trial

Pediatric Crohn's Disease (CD) is associated with low trabecular bone mineral density (BMD), cortical area, and muscle mass. Low‐magnitude mechanical stimulation (LMMS) may be anabolic. We conducted a 12‐month randomized double‐blind placebo‐controlled trial of 10 minutes daily exposure to LMMS (30 Hz frequency, 0.3 g peak‐to‐peak acceleration). The primary outcomes were tibia trabecular BMD and cortical area by peripheral quantitative CT (pQCT) and vertebral trabecular BMD by QCT; additional outcomes included dual‐energy X‐ray absorptiometry (DXA) whole body, hip and spine BMD, and leg lean mass. Results were expressed as sex‐specific Z‐scores relative to age. CD participants, ages 8 to 21 years with tibia trabecular BMD <25th percentile for age, were eligible and received daily cholecalciferol (800 IU) and calcium (1000 mg). In total, 138 enrolled (48% male), and 121 (61 active, 60 placebo) completed the 12‐month trial. Median adherence measured with an electronic monitor was 79% and did not differ between arms. By intention‐to‐treat analysis, LMMS had no significant effect on pQCT or DXA outcomes. The mean change in spine QCT trabecular BMD Z‐score was +0.22 in the active arm and –0.02 in the placebo arm (difference in change 0.24 [95% CI 0.04, 0.44]; p = 0.02). Among those with >50% adherence, the effect was 0.38 (95% CI 0.17, 0.58, p < 0.0005). Within the active arm, each 10% greater adherence was associated with a 0.06 (95% CI 0.01, 1.17, p = 0.03) greater increase in spine QCT BMD Z‐score. Treatment response did not vary according to baseline body mass index (BMI) Z‐score, pubertal status, CD severity, or concurrent glucocorticoid or biologic medications. In all participants combined, height, pQCT trabecular BMD, and cortical area and DXA outcomes improved significantly. In conclusion, LMMS was associated with increases in vertebral trabecular BMD by QCT; however, no effects were observed at DXA or pQCT sites. © 2016 American Society for Bone and Mineral Research.     

Nicotinamide phosphoribosyltransferase (Nampt) affects the lineage fate determination of mesenchymal stem cells: A possible cause for reduced osteogenesis and increased adipogenesis in older individuals

Human aging is associated with a progressive decline in bone mass and an accumulation of marrow fat. We found that osteoblast differentiation was reduced and adipocyte formation increased in bone marrow stromal cells derived from aged mice compared with young controls. The increased adipogenesis correlated with a relatively lower Sirt1 activity and a lower intracellular NAD⁺ concentration. We suppose that these effects were caused by age‐related reduction of nicotinamide phosphoribosyltransferase (Nampt), the enzyme catalyzing NAD resynthesis from nicotinamide (NAM). In support of this hypothesis, treatment with Nampt inhibitor FK866 increased adipocyte formation and reduced mineralization in primary cultured bone marrow stromal cells. In addition, knockdown of Nampt in the mouse mesenchymal cell line C3H10T1/2 cells resulted in decreased Sirt1 activity and enhanced adipogenesis. Interestingly, although Nampt deficiency resulted in both decreased intracellular NAD⁺ and increased NAM, the cell differentiation could be controlled only by regulation of NAM. These results indicate that the lineage fate determination of mesenchymal stem cells (MSCs) is influenced by cell energy metabolism and points to a possible mechanism for the development of senile osteoporosis. Furthermore, we suggest that side effects on bone should be considered when evaluating the long‐term safety of NAD‐interfering pharmaceuticals. © 2011 American Society for Bone and Mineral Research     

Defects in mesenchymal stem cell self‐renewal and cell fate determination lead to an osteopenic phenotype in Bmi‐1 null mice

In parathyroid hormone–related protein 1‐84 [PTHrP(1‐84)] knockin mice, expression of the polycomb protein Bmi‐1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi‐1^?/? mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM‐MSCs) from Bmi‐1^?/? mice ex vivo in culture. Neonatal Bmi‐1^?/? mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar‐γ expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1‐84) knockin mouse. The efficiency of colony‐forming unit fibroblast (CFU‐F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase–positive CFU‐F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar‐γ expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM‐MSCs were associated with upregulation of senescence‐associated tumor‐suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi‐1^?/? mice suggests that Bmi‐1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi‐1 maintains self‐renewal of BM‐MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM‐MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi‐1 therefore plays a critical role in promoting osteogenesis. © 2010 American Society for Bone and Mineral Research     

Two‐step stem cell therapy improves bone regeneration compared to concentrated bone marrow therapy

Adult stem cells are a promising tool to positively influence bone regeneration. Concentrated bone marrow therapy entails isolating osteoprogenitor cells during surgery with, however, only low cells yield. Two step stem cell therapy requires an additional harvesting procedure but generates high numbers of progenitor cells that facilitate osteogenic pre‐differentiation. To further improve bone regeneration, stem cell therapy can be combined with growth factors from platelet rich plasma (PRP) or its lysate (PL) to potentially fostering vascularization. The aim of this study was to investigate the effects of bone marrow concentrate (BMC), osteogenic pre‐differentiation of mesenchymal stromal cells (MSCs), and PL on bone regeneration and vascularization. Bone marrow from four different healthy human donors was used for either generation of BMC or for isolation of MSCs. Seventy‐two mice were randomized to six groups (Control, PL, BMC, BMC + PL, pre‐differentiated MSCs, pre‐differentiated MSCs + PL). The influence of PL, BMC, and pre‐differentiated MSCs was investigated systematically in a 2 mm femoral bone defect model. After a 6‐week follow‐up, the pre‐differentiated MSCs + PL group showed the highest bone volume, highest grade of histological defect healing and highest number of bridged defects with measurable biomechanical stiffness. Using expanded and osteogenically pre‐differentiated MSCs for treatment of a critical‐size bone defect was favorable with regards to bone regeneration compared to treatment with cells from BMC. The addition of PL alone had no significant influence; therefore the role of PL for bone regeneration remains unclear. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1318–1328, 2019.     

Sex‐Dependent,Osteoblast Stage‐Specific Effects of Progesterone Receptor on Bone Acquisition

The role of the progesterone receptor (PR) in the regulation of sexual dimorphism in bone has yet to be determined. Here we utilized genetic fate mapping and Western blotting to demonstrate age‐dependent PR expression in the mouse femoral metaphysis and diaphysis. To define sex‐dependent and osteoblast stage–specific effects of PR on bone acquisition, we selectively deleted PR at different stages of osteoblast differentiation. We found that when Prx1‐Cre mice were crossed with PR floxed mice to generate a mesenchymal stem cell (MSC) conditional KO model (Prx1; PRcKO), the mutant mice developed greater trabecular bone volume with higher mineral apposition rate and bone formation. This may be explained by increased number of MSCs and greater osteogenic potential, particularly in males. Age‐related trabecular bone loss was similar between the Prx1; PRcKO mice and their WT littermates in both sexes. Hormone deficiency during the period of rapid bone growth induced rapid trabecular bone loss in both the WT and the Prx1; PRcKO mice in both sexes. No differences in trabecular bone mass was observed when PR was deleted in mature osteoblasts using osteocalcin‐Cre (Bglap‐Cre). Also, there were no differences in cortical bone mass in all three PRcKO mice. In conclusion, PR inactivation in early osteoprogenitor cells but not in mature osteoblasts influenced trabecular bone accrual in a sex‐dependent manner. PR deletion in osteoblast lineage cells did not affect cortical bone mass. © 2017 American Society for Bone and Mineral Research.     

Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 ± 0.6 million and 10.5 ± 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri‐lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s‐GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s‐GAG expressions to chondrocytes. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:634–642, 2012     

Application of mesenchymal stromal cells in urological diseases

The application of stem cells and their use in tissue‐engineering approaches is emerging in clinical therapeutic intervention strategies. The use of adult stem cells, either autologous or allogenic, does not raise ethical concerns, in contrast to embryonic stem cells. Mesenchymal stromal cells (MSCs) can be easily obtained from bone marrow or from adipose tissue and further expanded in vitro. Due to their differentiation capacity, MSCs are very attractive for tissue engineering purposes. Furthermore, MSCs secrete a variety of mediators that have beneficial effects on the regenerating tissue. In this review we give an insight into stem cell hierarchy, define the properties of MSCs and summarize recent reports of their administration in urological diseases.     

Caloric restriction leads to high marrow adiposity and low bone mass in growing mice

The effects of caloric restriction (CR) on the skeleton are well studied in adult rodents and include lower cortical bone mass but higher trabecular bone volume. Much less is known about how CR affects bone mass in young, rapidly growing animals. This is an important problem because low caloric intake during skeletal acquisition in humans, as in anorexia nervosa, is associated with low bone mass, increased fracture risk, and osteoporosis in adulthood. To explore this question, we tested the effect of caloric restriction on bone mass and microarchitecture during rapid skeletal growth in young mice. At 3 weeks of age, we weaned male C57Bl/6J mice onto 30% caloric restriction (10% kcal/fat) or normal diet (10% kcal/fat). Outcomes at 6 (n = 4/group) and 12 weeks of age (n = 8/group) included body mass, femur length, serum leptin and insulin‐like growth factor 1 (IGF‐1) values, whole‐body bone mineral density (WBBMD, g/cm²), cortical and trabecular bone architecture at the midshaft and distal femur, bone formation and cellularity, and marrow fat measurement. Compared with the normal diet, CR mice had 52% and 88% lower serum leptin and 33% and 39% lower serum IGF‐1 at 6 and 12 weeks of age (p < .05 for all). CR mice were smaller, with lower bone mineral density, trabecular, and cortical bone properties. Bone‐formation indices were lower, whereas bone‐resorption indices were higher (p < .01 for all) in CR versus normal diet mice. Despite having lower percent of body fat, bone marrow adiposity was elevated dramatically in CR versus normal diet mice (p < .05). Thus we conclude that caloric restriction in young, growing mice is associated with impaired skeletal acquisition, low leptin and IGF‐1 levels, and high marrow adiposity. These results support the hypothesis that caloric restriction during rapid skeletal growth is deleterious to cortical and trabecular bone mass and architecture, in contrast to potential skeletal benefits of CR in aging animals. © 2010 American Society for Bone and Mineral Research.     

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

Adenovirus 36, adiposity,and bone strength in late‐adolescent females

Adenovirus 36 (Ad36) is the only adenovirus to date that has been linked with obesity in humans. Our previous studies in late‐adolescent females suggest that excess weight in the form of fat mass is associated with lower cortical bone strength. The purpose of this study was to assess the relationship between Ad36‐specific antibodies, adiposity, and bone strength in our sample of late‐adolescent females. A cross‐sectional study of 115 females aged 18 to 19 years was performed. Participants were classified according to adiposity by dual‐energy X‐ray absorptiometry (body fat percentage as normal‐fat [ < 32% body fat; n = 93] or high‐fat [ ≥ 32% body fat; n = 22]), and according to the presence of Ad36‐specific neutralizing antibodies. Peripheral quantitative computed tomography measured bone parameters at the 4% (trabecular bone) and 20% (cortical bone) site, and muscle cross‐sectional area (MCSA) at the 66% site, from the distal metaphyses of the radius and the tibia. Bone strength was determined from volumetric bone mineral density and bone geometry to calculate bone strength index (BSI; trabecular site) and polar strength–strain index (SSI; cortical site). After adjustment for MCSA and limb length, radial SSI was lower in Ad36+ versus Ad36– subjects from the high‐fat group (p < 0.03), but not the normal‐fat group. No significant differences were observed between groups in tibial SSI or BSI. These data support an association of adiposity and cortical bone strength at the radius with the presence of neutralizing antibodies to Ad36 in late‐adolescent females. © 2013 American Society for Bone and Mineral Research.     

Deficiency of Sef Is Associated With Increased Postnatal Cortical Bone Mass by Regulating Runx2 Activity

Sef (similar expression to fgf genes) is a feedback inhibitor of fibroblast growth factor (FGF) signaling and functions in part by binding to FGF receptors and inhibiting their activation. Genetic studies in mice and humans indicate an important role for fibroblast growth factor signaling in bone growth and homeostasis. We, therefore, investigated whether Sef had a function role in skeletal acquisition and remodeling. Sef expression is increased during osteoblast differentiation in vitro, and LacZ staining of Sef+/? mice showed high expression of Sef in the periosteum and chondro‐osseous junction of neonatal and adult mice. Mice with a global deletion of Sef showed increased cortical bone thickness, bone volume, and increased periosteal perimeter by micro‐computed tomography (micro‐CT). Histomorphometric analysis of cortical bone revealed a significant increase in osteoblast number. Interestingly, Sef?/? mice showed very little difference in trabecular bone by micro‐CT and histomorphometry compared with wild‐type mice. Bone marrow cells from Sef?/? mice grown in osteogenic medium showed increased proliferation and increased osteoblast differentiation compared with wild‐type bone marrow cells. Bone marrow cells from Sef?/? mice showed enhanced FGF2‐induced activation of the ERK pathway, whereas bone marrow cells from Sef transgenic mice showed decreased FGF2‐induced signaling. FGF2‐induced acetylation and stability of Runx2 was enhanced in Sef?/? bone marrow cells, whereas overexpression of Sef inhibited Runx2‐responsive luciferase reporter activity. Bone marrow from Sef?/? mice showed enhanced hematopoietic lineage‐dependent and osteoblast‐dependent osteoclastogenesis and increased bone resorptive activity relative to wild‐type controls in in vitro assays, whereas overexpression of Sef inhibited osteoclast differentiation. Taken together, these studies indicate that Sef has specific roles in osteoblast and osteoclast lineages and that its absence results in increased osteoblast and osteoclast activity with a net increase in cortical bone mass. © 2014 American Society for Bone and Mineral Research.     

Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells

Mesenchymal stem cells (MSCs), which can be isolated from bone marrow and other somatic tissues, are residing in an environment with relative low oxygen tension. The purpose of this study is to investigate the effects of hypoxia on MSCs, and we hypothesize that oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards differentiation. In this study, human bone marrow‐derived MSCs were cultured under hypoxia with 1% O₂. The proliferation ability of MSCs was increased after a 7‐day hypoxic culture period. Migration assay showed that hypoxia enhanced the migration capabilities of MSCs. Moreover, expression of stemness genes Oct4, Nanog, Sall4 and Klf4 was increased under hypoxia. Furthermore, the differentiation ability of MSCs under hypoxia favored osteogenesis while adipogenesis was inhibited during a 4‐week induction period. Cytokine antibody array analysis showed that a number of growth factors were up‐regulated after a 7‐day hypoxic incubation and the differential expression of growth factors may account for the increased proliferation and osteogenic potentials of MSCs under hypoxic condition. Taken together, hypoxia provides a favorable culture condition to promote proliferation as well as osteogenesis of MSCs through differential growth factor production. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:260–266, 2012     

Development and characterization of novel clinical grade neonatal porcine bone marrow‐derived mesenchymal stem cells

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.     

High‐Fat Diet–Induced Obesity Promotes Expansion of Bone Marrow Adipose Tissue and Impairs Skeletal Stem Cell Functions in Mice

Obesity represents a risk factor for development of insulin resistance and type 2 diabetes. In addition, it has been associated with increased adipocyte formation in the bone marrow (BM) along with increased risk for bone fragility fractures. However, little is known on the cellular mechanisms that link obesity, BM adiposity, and bone fragility. Thus, in an obesity intervention study in C57BL/6J mice fed with a high‐fat diet (HFD) for 12 weeks, we investigated the molecular and cellular phenotype of bone marrow adipose tissue (BMAT), BM progenitor cells, and BM microenvironment in comparison to peripheral adipose tissue (AT). HFD decreased trabecular bone mass by 29%, cortical thickness by 5%, and increased BM adiposity by 184%. In contrast to peripheral AT, BMAT did not exhibit pro‐inflammatory phenotype. BM progenitor cells isolated from HFD mice exhibited decreased mRNA levels of inflammatory genes (Tnfα, IL1β, Lcn2) and did not manifest an insulin resistant phenotype evidenced by normal levels of pAKT after insulin stimulation as well as normal levels of insulin signaling genes. In addition, BM progenitor cells manifested enhanced adipocyte differentiation in HFD condition. Thus, our data demonstrate that BMAT expansion in response to HFD exerts a deleterious effect on the skeleton. Continuous recruitment of progenitor cells to adipogenesis leads to progenitor cell exhaustion, decreased recruitment to osteoblastic cells, and decreased bone formation. In addition, the absence of insulin resistance and inflammation in the BM suggest that BMAT buffers extra energy in the form of triglycerides and thus plays a role in whole‐body energy homeostasis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.