Common Mesenchymal Stem Cells Research Articles

Bone Morphogenic Protein and Mesenchymal Stem Cells to Regenerate Bone in Calvarial Defects: A Systematic Review.

The use of bone morphogenic protein and mesenchymal stem cells has shown promise in promoting bone regeneration in calvarial defects. However, a systematic review of the available literature is needed to evaluate the efficacy of this approach. We comprehensively searched electronic databases using MeSH terms related to skull defects, bone marrow mesenchymal stem cells, and bone morphogenic proteins. Eligible studies included animal studies that used BMP therapy and mesenchymal stem cells to promote bone regeneration in calvarial defects. Reviews, conference articles, book chapters, and non-English language studies were excluded. Two independent investigators conducted the search and data extraction. Twenty-three studies published between 2010 and 2022 met our inclusion criteria after a full-text review of the forty-five records found in the search. Eight of the 23 studies used mice as models, while 15 used rats. The most common mesenchymal stem cell was bone marrow-derived, followed by adipose-derived. BMP-2 was the most popular. Stem cells were embedded in Scaffold (13), Transduction (7), and Transfection (3), and they were delivered BMP to cells. Each treatment used 2 × 104-1 × 107 mesenchymal stem cells, averaging 2.26 × 106. Most BMP-transduced MSC studies used lentivirus. This systematic review examined BMP and MSC synergy in biomaterial scaffolds or alone. BMP therapy and mesenchymal stem cells in calvarial defects, alone, or with a scaffold regenerated bone. This method treats skull defects in clinical trials. The best scaffold material, therapeutic dosage, administration method, and long-term side effects need further study.

Cardiac Progenitor Cells and Adipocyte Stem Cells from Same Patients Exhibit In Vitro Functional Differences.

Cardiac progenitor cells (CPCs) and adipocyte stem cells (ASCs) are widely tested for their efficacy in repairing the diseased heart with varying results. However, no study has directly compared the functional efficacy of CPCs and ASCs collected from the same patient. CPCs and ASCs were isolated from the right atrial appendage and epicardial adipose tissue of the same patients, using explant culture. The flow cytometry analysis confirmed that both the cell types express common mesenchymal stem cells markers CD90 and CD105. ASCs, in addition, expressed CD29 and CD73. The wound-healing assay demonstrated that CPCs migrate faster to cover the wound area. Both cell types were resistant to hypoxia-induced cell death when exposed to hypoxia and serum deprivation; however, the ASCs showed increased proliferation. Conditioned medium (CM) collected after culturing serum-deprived CPCs and ASCs showed differential secretion patterns, with ASC CM showing an increased IGF-1 level, while CPC CM showed an increased FGF level. Only CPC CM reduced hypoxia-induced apoptosis in AC-16 human ventricular cardiomyocytes, while vascular network formation by endothelial cells was comparable between CPC and ASC CM. In conclusion, ASCs and CPCs exhibit differential characteristics within the same patient, and in vitro studies showed that CPCs have marginally superior functional efficacy.

A comparative study of the capability of MSCs isolated from different human tissue sources to differentiate into neuronal stem cells and dopaminergic-like cells.

BackgroundNeurodegenerative diseases are characterized by progressive neuronal loss and degeneration. The regeneration of neurons is minimal and neurogenesis is limited only to specific parts of the brain. Several clinical trials have been conducted using Mesenchymal Stem Cells (MSCs) from different sources to establish their safety and efficacy for the treatment of several neurological disorders such as Parkinson’s disease, multiple sclerosis and amyotrophic lateral sclerosis.AimThe aim of this study was to provide a comparative view of the capabilities of MSCs, isolated from different human tissue sources to differentiate into neuronal stem cell-like cells (NSCs) and possibly into dopaminergic neural- like cells.MethodsMesenchymal stem cells were isolated from human bone marrow, adipose, and Wharton’s jelly (WJ) tissue samples. Cells were characterized by flow cytometry for their ability to express the most common MSC markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of these cells to differentiate towards the neural stem cell-like lineage, cells were cultured in media containing small molecules. Cells were utilized for gene expression and immunofluorescence analysis at different time points.ResultsOur results indicate that we have successfully isolated MSCs from bone marrow, adipose tissue, and Wharton’s jelly. WJ-MSCs showed a slightly higher proliferation rate after 72 hours compared to BM and AT derived MSCs. Gene expression of early neural stem cell markers revealed that WJ-MSCs had higher expression of Nestin and PAX6 compared to BM and AT-MSCs, in addition to LMX expression as an early dopaminergic neural marker. Immunofluorescence analysis also revealed that these cells successfully expressed SOX1, SOX2, Nestin, TUJ1, FOXA2 and TH.ConclusionThese results indicate that the protocol utilized has successfully differentiated BM, AT and WJ-MSCs into NSC-like cells. WJ-MSCs possess a higher potential to transdifferentiate into NSC and dopaminergic-like cells. Thus, it might indicate that this protocol can be used to induce MSC into neuronal lineage, which provides an additional or alternative source of cells to be used in the neurological cell-based therapies.

Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity.

BackgroundThe heterogeneity of mesenchymal stem cells (MSCs) is poorly understood, thus limiting clinical application and basic research reproducibility. Advanced single‐cell RNA sequencing (scRNA‐seq) is a robust tool used to analyse for dissecting cellular heterogeneity. However, the comprehensive single‐cell atlas for human MSCs has not been achieved.MethodsThis study used massive parallel multiplexing scRNA‐seq to construct an atlas of > 130 000 single‐MSC transcriptomes across multiple tissues and donors to assess their heterogeneity. The most widely clinically utilised tissue resources for MSCs were collected, including normal bone marrow (n = 3), adipose (n = 3), umbilical cord (n = 2), and dermis (n = 3).ResultsSeven tissue‐specific and five conserved MSC subpopulations with distinct gene‐expression signatures were identified from multiple tissue origins based on the high‐quality data, which has not been achieved previously. This study showed that extracellular matrix (ECM) highly contributes to MSC heterogeneity. Notably, tissue‐specific MSC subpopulations were substantially heterogeneous on ECM‐associated immune regulation, antigen processing/presentation, and senescence, thus promoting inter‐donor and intra‐tissue heterogeneity. The variable dynamics of ECM‐associated genes had discrete trajectory patterns across multiple tissues. Additionally, the conserved and tissue‐specific transcriptomic‐regulons and protein‐protein interactions were identified, potentially representing common or tissue‐specific MSC functional roles. Furthermore, the umbilical‐cord‐specific subpopulation possessed advantages in immunosuppressive properties.ConclusionIn summary, this work provides timely and great insights into MSC heterogeneity at multiple levels. This MSC atlas taxonomy also provides a comprehensive understanding of cellular heterogeneity, thus revealing the potential improvements in MSC‐based therapeutic efficacy.

Deficiency of PPARγ in Bone Marrow Stromal Cells Does not Prevent High-Fat Diet-Induced Bone Deterioration in Mice

BackgroundBone marrow osteoblasts and adipocytes are derived from a common mesenchymal stem cell and have a reciprocal relationship. Peroxisome proliferator-activated receptor gamma (PPARγ), a regulator for adipocyte differentiation, may be a potential target for reducing obesity and increasing bone mass. ObjectivesThis study tested the hypothesis that bone-specific Pparg conditional knockout (cKO), via deletion of Pparg from bone marrow stromal cells (BMSC) using Osterix 1 (Osx1)-Cre, would prevent high-fat (HF) diet–induced bone deterioration in mice. MethodsPPARγ cKO (PPARγfl/fl: Osx1-Cre) and floxed littermate control (PPARγfl/fl Osx1-Cre–) mice that were 6 weeks old were randomly assigned to 4 groups (n = 12/group, 6 male and 6 female) and fed ad libitum with either a normal-fat (NF) purified diet (3.85 kcal/g; 10% energy as fat) or an HF diet (4.73 kcal/g; 45% energy as fat) for 6 mo. Bone structure, body composition, and serum bone-related cytokines were measured. Data were analyzed by 2-way ANOVA with Tukey post hoc comparison. ResultsThe HF diet decreased the tibial and lumbar vertebrae trabecular bone volume/total volume (BV/TV) by 28% and 18%, respectively, compared to the NF diet (P < 0.01). PPARγ cKO mice had 23% lower body fat mass and 9% lower lean mass than control mice. PPARγ cKO mice had 41% greater tibial trabecular BV/TV compared to control mice. None of trabecular bone parameters at the second lumbar vertebra were affected by genotype. PPARγ cKO mice had decreased cortical thickness compared to control mice. PPARγ cKO mice had a 14% lower (P < 0.01) serum concentration of leptin and a 35% higher (P < 0.05) concentration of osteocalcin compared with control mice. ConclusionsThese data indicate that PPARγ has site-specific impacts on bone structures in mice and that knockout PPARγ in BMSC increased bone mass (BV/TV) in the tibia but not the lumbar vertebrae. PPARγ disruption in BMSC did not prevent HF diet–induced bone deterioration in mice.

Deficiency of PPAR Gamma in Bone Marrow Stromal Cells Does Not Prevent High-Fat Diet-Induced Bone Deterioration in Mice

ObjectivesObesity is detrimental to bone health in humans and in high-fat diet-induced obese animals. Bone marrow osteoblasts and adipocytes are derived from a common mesenchymal stem cell and have a reciprocal relationship. Peroxisome proliferator-activated receptor gamma (PPAR gamma), a regulator for adipocyte differentiation, may be a potential target for reducing obesity and increasing bone mass. This study tested the hypothesis that bone-specific PPAR gamma conditional knockout (cKO), via deletion of PPAR gamma gene from bone marrow stromal cells (BMSC) using Osterix 1 (Osx1)-Cre, would prevent high-fat diet-induced bone deterioration in mice. MethodsPPAR gamma cKO (PPAR gammafl/fl: Osx1–Cre) and floxed littermate control (PPAR gammafl/fl Osx1-P-Cre-) mice at 6-wk-old were randomly assigned to 4 groups (n = 12/group, 6 male and 6 female) and fed ad libitum either a normal-fat purified diet (NF, 3.85 kcal/g, 10% energy as fat) or a high-fat diet (HF, 4.73 kcal/g) for 6 mo. Bone structure, body composition, and serum bone-related cytokines were measured. ResultsCompared to the NF diet, the HF diet increased body mass and fat mass (P < 0.05) but not lean mass. The HF diet also decreased tibial and lumbar vertebrae trabecular volume/total volume (BV/TV) and bone mineral density (BMD) in both control and PPAR gamma cKO mice. PPAR gamma cKO mice had lower body fat mass and lean mass than control mice. PPAR gamma cKO mice had greater tibial trabecular BV/TV, trabecular number, connectivity density, and BMD and lower structure model index, compared to control mice. None of trabecular bone parameters at 2nd lumbar vertebrae was affected by genotype. PPAR gamma cKO mice had lower cortical medullary area, compared to control mice. PPAR gamma cKO mice had lower (P < 0.01) serum concentration of leptin and higher (P < 0.05) concentration of osteocalcin, compared with control mice. ConclusionsThese data indicate that PPAR gamma has site-specific impact on bone structure in mice and that knockout PPAR gamma in BMSC increased bone mass in tibia likely through increased osteoblastogenesis. However, PPAR gamma disruption in BMSC did not prevent high-fat diet-induced bone deterioration in mice. Funding SourcesUSDA-ARS #3062-51,000-053-00D.

Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells.

BackgroundCongenital abnormalities, cancers as well as injuries can cause irreversible damage to the urinary tract, which eventually requires tissue reconstruction. Smooth muscle cells, endothelial cells, and urothelial cells are the major cell types required for the reconstruction of lower urinary tract. Adult stem cells represent an accessible source of unlimited repertoire of untransformed cells.AimFetal bovine serum (FBS) is the most vital supplement in the culture media used for cellular proliferation and differentiation. However, due to the increasing interest in manufacturing xeno-free stem cell-based cellular products, optimizing the composition of the culture media and the serum-type used is of paramount importance. In this study, the effects of FBS and pooled human platelet (pHPL) lysate were assessed on the capacity of human adipose-derived stem cells (ADSCs) to differentiate into urothelial-like cells. Also, we aimed to compare the ability of both conditioned media (CM) and unconditioned urothelial cell media (UCM) to induce urothelial differentiation of ADCS in vitro.MethodsADSCs were isolated from human lipoaspirates and characterized by flow cytometry for their ability to express the most common mesenchymal stem cell (MSCs) markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of ADSCs to differentiate towards the urothelial-like lineage, cells were cultured with either CM or UCM, supplemented with either 5% pHPL, 2.5% pHPL or 10% FBS. After 14 days of induction, cells were utilized for gene expression and immunofluorescence analysis.ResultsADSCs cultured in CM and supplemented with FBS exhibited the highest upregulation levels of the urothelial cell markers; cytokeratin-18 (CK-18), cytokeratin-19 (CK-19), and Uroplakin-2 (UPK-2), with a 6.7, 4.2- and a 2-folds increase in gene expression, respectively. Meanwhile, the use of CM supplemented with either 5% pHPL or 2.5% pHPL, and UCM supplemented with either 5% pHPL or 2.5% pHPL showed low expression levels of CK-18 and CK-19 and no upregulation of UPK-2 level was observed. In contrast, the use of UCM with FBS has increased the levels of CK-18 and CK-19, however to a lesser extent compared to CM. At the cellular level, CK-18 and UPK-2 were only detected in CM/FBS supplemented group. Growth factor analysis revealed an increase in the expression levels of EGF, VEGF and PDGF in all of the differentiated groups.ConclusionEfficient ADSCs urothelial differentiation is dependent on the use of conditioned media. The presence of high concentrations of proliferation-inducing growth factors present in the pHPL reduces the efficiency of ADSCs differentiation towards the urothelial lineage. Additionally, the increase in EGF, VEGF and PDGF during the differentiation implicates them in the mechanism of urothelial cell differentiation.

Autologous mesenchymal stem cells application in post-burn scars treatment: a preliminary study

Mesenchymal stem cells (MSCs) are multi-potent cells characterized by long term self-renewal and by potential for differentiation into cells of different mesenchymal tissue types such as fibroblasts, osteocytes, chondrocytes, and adipocytes. Their unique properties offer broad therapeutic potentials. Bone marrow has been used as the most common MSCs source, but it is gradually going to be replaced by adipose tissue which showed to contain more MSCs per unit than the bone marrow and clinical application of MSCs procured from the adipose tissue have been demonstrating at least similar results. Post-burn scars result frequently in severe both functional and aesthetic impairments in restitution and rehabilitation periods of the burn disease. Despite extensive research in the last decades, the exact mechanisms of scar formation remains unclear. The development of post-burn scars is influenced by multiple factors such as initial depth of the burn, methods of burn wound therapy, duration of the open wound until final wound closure, burn wound infection, genetic predisposition, and many others in both acute and rehabilitation periods. The aim of this study was to point out versatility of the implementation of this method with respect to different types of scars (atrophic scars, hypertrophic scars, keloids). Autologous adipose tissue derived MSCs were applied to post-burn scars in all 8 patients undergoing surgical scar reconstructions at the Department of Burns and Reconstructive Surgery of the University Hospital in Bratislava. The study was approved by Ethical Committee of Ruzinov Hospital. The procedures used for scar reconstructions included dermabrasion, scar excisions, contractures corrections and local plasties combined by lipografting of lipoaspirate containing parenchymal adipocytes and stromal vascular fraction including MSCs, or application of separated autologous MSCs isolated from lipoaspirates. Based on desired result one of these MSCs application methods was selected depending on characteristics of reconstructed scar and required volume of transferred fat. Isolation of MSCs following procurement was provided by the Central Tissue Bank cell culture laboratory which is one of the parts of the burn department. The average time of scars duration was 79months, ranging from 6 to 216months. The postburns scars were assessed clinically according to Vancouver Scar Scale (VSS) prior to surgery, including photo documentation, and re-evaluated after 6months following MSCs application. As the results have shown, the average VSS score before treatment was 7.88 points ranging from 4 to 11 points. The average VSS 6months after surgical procedure and MSCs application was 2.34 points ranging from 1 to 4 points. According to the results obtained, the favourable effect of adipose tissue derived autologous MSCs application on scar remodelling following surgical reconstruction of post-burn scars could be promising.

Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties.

Can endometrial stromal stem/progenitor cell markers, SUSD2 and CD146/CD140b, enrich for human myometrial and fibroid stem/progenitor cells? SUSD2 enriches for myometrial and fibroid cells that have mesenchymal stem cell (MSC) characteristics and can also be induced to decidualise. Mesenchymal stem-like cells have been separately characterised in the endometrial stroma and myometrium and may contribute to diseases in their respective tissues. Normal myometrium, fibroids and endometrium were collected from hysterectomies with informed consent. Primary cells or tissues were used from at least three patient samples for each experiment. Flow cytometry, immunohistochemistry and immunofluorescence were used to characterise tissues. In vitro colony formation in normoxic and hypoxic conditions, MSC lineage differentiation (osteogenic and adipogenic) and decidualisation were used to assess stem cell activity. Xenotransplantation into immunocompromised mice was used to determine in vivo stem-like activity. Endpoint measures included quantitative PCR, colony formation, trichrome, Oil Red O and alkaline phosphatase activity staining. CD146+CD140b+ and/or SUSD2+ myometrial and fibroid cells were located in the perivascular region and formed more colonies in vitro compared to control cells and differentiated down adipogenic and osteogenic mesenchymal lineages in vitro. SUSD2+ myometrial cells had greater in vitro decidualisation potential, and SUSD2+ fibroid cells formed larger tumours in vivo compared to control cells. N/A. Markers used in this study enrich for cells with stem/progenitor cell activity; however, they do not distinguish stem from progenitor cells. SUSD2+ myometrial cells express markers of decidualisation when treated in vitro, but in vivo assays are needed to fully demonstration their ability to decidualise. These results suggest a possible common MSC for the endometrial stroma and myometrium, which could be the tumour-initiating cell for uterine fibroids. These studies were supported by NIH grants to JMT (R01OD012206) and to ALP (F32HD081856). The authors certify that we have no conflicts of interest to disclose.

Immunophenotypic expression of UCP1 in hibernoma and other adipose/non adipose soft tissue tumours

BackgroundUncoupling protein 1 (UCP1) is a mitochondral protein transporter that uncouples electron transport from ATP production. UCP1 is highly expressed in brown adipose tissue (BAT), including hibernomas, but its expression in other adipose tumours is uncertain. UCP1 has also been found in other tissues (e.g. smooth muscle) but whether it is expressed in non-adipose benign and malignant soft tissue tumours is unknown.MethodsImmunohistochemical staining of normal (axillary) BAT and subcutaneous/abdominal white adipose tissue (WAT) as well as a wide range of benign and malignant primary soft tissue tumours (n = 171) was performed using a rabbit polyclonal antibody to UCP1. BAT and hibernomas were also stained by immunohistochemistry with monoclonal and polyclonal antibodies to adipose/non-adipose tumour markers in order to characterise the immunophenotype of BAT cells.ResultsUCP1 was strongly expressed in the cytoplasm of brown fat cells in BAT and hibernomas, both of which also expressed aP2, S100, CD31, vimentin and calponin. UCP1 was not expressed in WAT or other adipose tumours with the exception a few tumour cells in pleomorphic liposarcoma. UCP1 was variably expressed by tumour cells in a few non-adipose sarcomas including leiomyosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, synovial sarcoma and clear cell sarcoma.ConclusionsUCP1 is strongly expressed in BAT but not WAT and is found in all hibernomas and a few pleomorphic liposarcomas but not in other adipose tumours. UCP1 expression in a few non-adipose soft tissue sarcomas may possibly reflect origin of tumour cells from a common mesenchymal stem cell precursor and/or developmental pathway.

Functional and Genomic Characterization of the Interaction between Acute Lymphoblastic Leukemia Cells and the Microenvironment Identifies Pathways for Therapeutic Intervention

IntroductionResidence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable.Materials and methodsTime-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence.ResultsTime-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%).Analysis of gene expression showed 138 upregulated genes (log2FC >2 and FDR <0.05) in adherent cells which were common in all five cell lines, with striking upregulation leading to gene expression associated with gene ontology of extracellular matrix organization and collagen fibril organization. Representative genes were validated in adherent NALM6 cells by immunoblotting (FN1, TIMP1, and LGALS1).Pathway analysis showed that transforming growth factor beta 1 (TGFB1) was the top ranked upstream regulator (p-value of overlap 5.26E-35) in that 44 of 65 genes had measurement direction consistent with activation of TGFB1 signaling. In addition, other upstream regulators that may be involved were beta-estradiol, fibroblast growth factor 2, and tumor necrosis factor.Adhesion of leukemia cells to stroma may induce integrin expression and downstream signaling. Our transcriptomic analysis showed that integrins (A5, B1, A3 and B5) and caveolin 1 (CAV1), a main component of the caveolae plasma membranes, are highly transcribed in adherent leukemia cells. As hTERT cells showed unexpectedly low expression of THY1, a common MSC marker, we utilized primary bone marrow MSC for subsequent analysis. Immunoblotting assay showed enhanced expression of CAV1 in all cell lines adhered to MSCs. Multicolor immunofluorescence imaging demonstrated CAV1 and ITGB1 expression on leukemia cells that localized adjacent to stromal cells, which confirms that these molecules were upregulated upon adhesion to MSCs. Moreover, primary ALL cells showed remarkable upregulation of CAV1, ITGB1, ITGA3, and ITGB5 when the cells were adherent to MSCs.ConclusionsOur results demonstrate that ALL cells dynamically interact with microenvironment cells, inducing changes in cell morphology, cell cycling, and adhesion, which may facilitate altered responsiveness to therapy. Transcriptional results suggest that TGFβ signaling is an upstream regulator after cell adhesion to MSCs. While integrins and CAV1 mediate the signaling, these pathway and molecules will be candidates for exploring inhibitors of signaling, which may affect their interaction and make them novel therapeutic targets. [Display omitted] DisclosuresMullighan:Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

MECHANISMS IN ENDOCRINOLOGY: Bone marrow adiposity and bone, a bad romance?

Bone marrow adipocytes (BMA-) constitute an original and heterogeneous fat depot whose development appears interlinked with bone status throughout life. The gradual replacement of the haematopoietic tissue by BMA arises in a well-ordered way during childhood and adolescence concomitantly to bone growth and continues at a slower rate throughout the adult life. Importantly, BM adiposity quantity is found well associated with bone mineral density (BMD) loss at different skeletal sites in primary osteoporosis such as in ageing or menopause but also in secondary osteoporosis consecutive to anorexia nervosa. Since BMA and osteoblasts originate from a common mesenchymal stem cell, adipogenesis is considered as a competitive process that disrupts osteoblastogenesis. Besides, most factors secreted by bone and bone marrow cells (ligands and antagonists of the WNT/β-catenin pathway, BMP and others) reciprocally regulate the two processes. Hormones such as oestrogens, glucocorticoids, parathyroid and growth hormones that control bone remodelling also modulate the differentiation and the activity of BMA. Actually, BMA could also contribute to bone loss through the release of paracrine factors altering osteoblast and/or osteoclast formation and function. Based on clinical and fundamental studies, this review aims at presenting and discussing these current arguments that support but also challenge the involvement of BMA in the bone mass integrity.

Fat-bone interaction within the bone marrow milieu: Impact on hematopoiesis and systemic energy metabolism.

The relationship between fat, bone and systemic metabolism is a growing area of scientific interest. Marrow adipose tissue is a well-recognized component of the bone marrow milieu and is metabolically distinct from current established subtypes of adipose tissue. Despite recent advances, the functional significance of marrow adipose tissue is still not clearly delineated. Bone and fat cells share a common mesenchymal stem cell (MSC) within the bone marrow, and hormones and transcription factors such as growth hormone, leptin, and peroxisomal proliferator-activated receptor γ influence MSC differentiation into osteoblasts or adipocytes. MSC osteogenic potential is more vulnerable than adipogenic potential to radiation and chemotherapy, and this confers a risk for an abnormal fat-bone axis in survivors following cancer therapy and bone marrow transplantation. This review provides a summary of data from animal and human studies describing the relationship between marrow adipose tissue and hematopoiesis, bone mineral density, bone strength, and metabolic function. The significance of marrow adiposity in other metabolic disorders such as osteoporosis, diabetes mellitus, and estrogen and growth hormone deficiency are also discussed. We conclude that marrow adipose tissue is an active endocrine organ with important metabolic functions contributing to bone energy maintenance, osteogenesis, bone remodeling, and hematopoiesis. Future studies on the metabolic role of marrow adipose tissue may provide the critical insight necessary for selecting targeted therapeutic interventions to improve altered hematopoiesis and augment skeletal remodeling in cancer survivors.

Marrow adipose tissue imaging in humans.

Bone strength is affected not only by bone mineral density (BMD) and bone microarchitecture but also its microenvironment. Recent studies have focused on the role of marrow adipose tissue (MAT) in the pathogenesis of bone loss. Osteoblasts and adipocytes arise from a common mesenchymal stem cell within bone marrow and many osteoporotic states, including aging, medication use, immobility, over - and undernutrition are associated with increased marrow adiposity. Advancements in imaging technology allow the non-invasive quantification of MAT. This article will review magnetic resonance imaging (MRI)- and computed tomography (CT)-based imaging technologies to assess the amount and composition of MAT. The techniques that will be discussed are anatomic T1-weighted MRI, water-fat imaging, proton MR spectroscopy, single energy CT and dual energy CT. Clinical applications of MRI and CT techniques to determine the role of MAT in patients with obesity, anorexia nervosa, and type 2 diabetes will be reviewed.

The Impact of Lipid Types and Liposomal Formulations on Osteoblast Adiposity and Mineralization.

Recent studies have demonstrated that fat accumulation in bone cells is detrimental to bone mass. Both adipocytes and osteoblasts are derived from common multipotent mesenchymal stem cells (MSCs) and hence the presence of fat may increase adipocyte proliferation, differentiation and fat accumulation while inhibiting osteoblast differentiation and bone formation. Lipids are common constituents in supramolecular vesicles (e.g., micelles or liposomes) that serve as drug delivery systems. Liposomal formulations such as Meriva® were proven to decrease joint pain and improve joint function in osteoarthritis (OA) patients. In this study, we evaluated how lipid types and liposomal formulations affect osteoblast behavior including cell viability, differentiation, mineralization and inflammation. Various liposomal formulations were prepared using different types of lipids, including phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE), cholesterol (Chol), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl), and 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) to investigate the impact on osteoblast differentiation and inflammation. The results indicated that cationic lipids, DC-cholesterol and DOTAP, presented higher dose-dependent cytotoxicity and caused high level of inflammatory responses. Due to the natural properties of lipids, all the lipids can induce lipid droplet formation in osteoblasts but the level of lipid droplet accumulation was different. In comparison with cationic lipids, neutral lipids induced less adiposity, and maintained high osteoblast mineralization. Similar to previous researches, we also confirmed an inverse relationship between lipid droplet formation and osteoblast mineralization in 7F2 mouse osteoblasts. Importantly, PC containing liposomes (PC only and PC/DOTAP) suppressed IL-1β-induced gene expression of COX-2 and MMP-3 but not Chol/DOTAP liposomes or DC-Chol/DOPE liposomes. Taken together, we suggested that PC contained liposomes could provide the best liposomal formulation for the treatment of bone diseases.

One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells.

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

Chemokine-Like Receptor 1 Is a Novel Wnt Target Gene that Regulates Mesenchymal Stem Cell Differentiation.

Bone remodeling is a dynamic process requiring the coordinated action of formative (osteoblast) and resorptive (osteoclast) cell populations. An imbalance of the development and function of these cell types underlies several chronic bone loss disorders such as osteoporosis. Increased bone marrow adipocyte numbers commonly occur with bone loss disorders and numerous studies have documented an inverse relationship between bone marrow fat and bone formation. Osteoblasts and adipocytes derive in a competitive fashion from a common mesenchymal stem cell (MSC) precursor. Generally, factors that promote MSC adipogenesis inhibit osteoblastogenesis and thereby, reduce bone formation. Previously we established that the secreted protein chemerin regulates adipogenic and osteoblastogenic differentiation of MSCs by signaling through chemokine-like receptor 1 (CMKLR1). However, the fundamental mechanisms by which chemerin/CMKLR1 influences lineage determination remain largely uncharacterized. Herein, we provide experimental evidence that chemerin/CMKLR1 regulates canonical Wnt signaling in MSCs by influencing the expression, subcellular location, and transcriptional activity of the central Wnt transducer, β-catenin. Moreover, we provide evidence that CMKLR1 is a novel Wnt responsive gene that functions in a negative feedback loop to limit osteoblastogenic Wnt signaling. Mechanistically, this entails Notch-dependent changes in the expression and function of key adipogenic and osteoblastogenic transcription factors, cell cycle proteins and chromatin remodeling enzymes. Consistent with this, MSCs from CMKLR1 knockout (-/-) mice exhibited similar dependency on Notch signaling to maintain osteoblastogenic differentiation. Taken together, our findings support a fundamental biological function for chemerin/CMKLR1 to balance osteoblastogenic and adipogenic signaling and thereby contribute to the maintenance of pluripotency in MSCs. Stem Cells 2017;35:711-724.

Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ - update and future development.

The efficacy of tendon-derived stem cells (TDSCs) for the promotion of tendon and tendon-bone junction repair has been reported in animal studies. Modulation of the tendon stem cell niche in vivo has also been reported to influence tendon structure. There is a need to have specific and reliable markers that can define TDSCs in vitro and tendon stem cells in situ for several reasons: to understand the basic biology of TDSCs and their subpopulations in vitro; to understand the identity, niches and functions of tendon/progenitor stem cells in vivo; to meet the governmental regulatory requirements for quality of TDSCs when translating the exciting preclinical findings into clinical trial/practice; and to develop new treatment strategies for mobilizing endogenous stem/progenitor cells in tendon. TDSCs were reported to express the common mesenchymal stem cell (MSC) markers and some embryonic stem cell (ESC) markers, and there were attempts to use these markers to label tendon stem cells in situ. Are these stem cell markers useful for the identification of TDSCs in vitro and tracking of tendon stem cells in situ? This review aims to discuss the values of the panel of MSC, ESC and tendon-related markers for the identification of TDSCs in vitro. Important factors influencing marker expression by TDSCs are discussed. The usefulness and limitations of the panel of MSC, ESC and tendon-related markers for tracking stem cells in tendon, especially tendon stem cells, in situ are then reviewed. Future research directions are proposed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0097-y) contains supplementary material, which is available to authorized users.