Mesenchymal Stem And Progenitor Cells Research Articles

Angelica sinensis polysaccharides ameliorated 5-Fluorouracil-induced damage of early B cell progenitors by alleviating oxidative stress of IL-7 producing mesenchymal stem and progenitor cells

B-lymphocytopenia among myelosuppression is the most intractable side effect of chemotherapy. Here, we investigated ways to alleviate 5-fluorouracil-caused stress hematopoietic impairment. We found that intraperitoneally injected ASP (Angelica sinensis polysaccharides) (100 mg/kg per day), one main active ingredient of Angelica sinensis, for consecutive 7 days, significantly recovered mouse bone marrow pro-B and pre-B cells, reversed the capacity of CFU-PreB colony forming, thus alleviating B cell reduction in the spleen and peripheral blood, as well as ameliorating immunoglobin from spleen and serum. The mechanism is related to the protective effects of ASP on IL-7 producing cells, including perivascular Leptin+ and CXCL12+ mesenchymal stem and progenitor cells (MSPCs), thus promoting IL-7 production, and activating IL-7R-mediated STAT5, PI3K-AKT signaling, including survival signals and EBF1, PAX5 transcription factor expression. Additionally, ASP’s IL-7 promoting effect was demonstrated to be associated with maintaining osteogenesis/adipogenesis balance of MSPCs via the NRF2 antioxidant pathway. Collectively, our findings indicate that ASP reverse stress B-lymphocytopenia via improving Nrf2 signaling, promoting IL-7 production in MSPCs, and subsequently maintaining survival, proliferation, and differentiation of B cell progenitors, which may represent a promising therapeutic strategy.

Improved cell adhesion to activated vapor silanization-biofunctionalized Ti-6Al-4V surfaces with ECM-derived oligopeptides

Titanium implants are widely used in traumatology and various orthopedic fields. Titanium and other metallic-based implants have limited structural and functional integration into the body, which translates into progressive prosthesis instability and the need for new surgical interventions that have enormous social and economic impacts. To enhance the biocompatibility of titanium implants, numerous biofunctionalization strategies have been developed. However, the problem persists, as more than 70% of implant failures are due to aseptic loosening. In this study we addressed the problem of improving the physiological engraftability and acceptability of titanium-based implants by applying a robust and versatile functionalization method based on the covalent immobilization of extracellular matrix (ECM)-derived oligopeptides on Ti-6Al-4V surfaces treated by activated vapor silanization (AVS). The feasibility of this technique was evaluated with two oligopeptides of different structures and compositions. These oligopeptides were immobilized on Ti-6Al-4V substrates by a combination of AVS and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The immobilization was shown to be stable and resistant to chemical denaturing upon sodium dodecyl sulfate treatment. On Ti-6Al-4V surfaces both peptides increased the attachment, spreading, rearrangement and directional growth of mesenchymal stem and progenitor cells (MSC) with chondro- and osteo-regenerative capacities. We also found that this biofunctionalization method (AVS-EDC/NHS) increased the attachment capacity of an immortalized cell line of neural origin with poor adhesive properties, highlighting the versatility and robustness of this method in terms of potential oligopeptides that may be used, and cell lineages whose anchorage to the biomaterial may be enhanced. Collectively, this novel functionalization strategy can accelerate the development of advanced peptide-functionalized metallic surfaces, which, in combination with host or exogenously implanted stem cells, have the potential to positively affect the osteoregenerative and osteointegrative abilities of metallic-based prostheses.

Mesenchymal stromal cells in the bone marrow niche consist of multi-populations with distinct transcriptional and epigenetic properties

Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

Surface Modifications of Titanium Aluminium Vanadium Improve Biocompatibility and Osteogenic Differentiation Potential.

Osteogenic cells are strongly influenced in their behaviour by the surface properties of orthopaedic implant materials. Mesenchymal stem and progenitor cells (MSPCs) migrate to the bone–implant interface, adhere to the material surface, proliferate and subsequently differentiate into osteoblasts, which are responsible for the formation of the bone matrix. Five surface topographies on titanium aluminium vanadium (TiAl6V4) were engineered to investigate biocompatibility and adhesion potential of human osteoblasts and the changes in osteogenic differentiation of MSPCs. Elemental analysis of TiAl6V4 discs coated with titanium nitride (TiN), silver (Ag), roughened surface, and pure titanium (cpTi) surface was analysed using energy-dispersive X-ray spectroscopy and scanning electron microscopy. In vitro cell viability, cytotoxicity, adhesion behaviour, and osteogenic differentiation potential were measured via CellTiter-Glo, CytoTox, ELISA, Luminex® technology, and RT-PCR respectively. The Ag coating reduced the growth of osteoblasts, whereas the viability of MSPCs increased significantly. The roughened and the cpTi surface improved the viability of all cell types. The additive coatings of the TiAl6V4 alloy improved the adhesion of osteoblasts and MSPCs. With regard to the osteogenic differentiation potential, an enhanced effect has been demonstrated, especially in the case of roughened and cpTi coatings.

Hematopoietic Stem Cell Niches and Signals Controlling Immune Cell Development and Maintenance of Immunological Memory.

Studies over the last couple of decades have shown that hematopoietic stem cells (HSCs) are critically dependent on cytokines such as Stem Cell Factor and other signals provided by bone marrow niches comprising of mesenchymal stem and progenitor cells (MSPCs) and endothelial cells (ECs). Because of their critical roles in HSC maintenance the niches formed by MSPCs and ECs are commonly referred to as HSC niches. For the most part, the signals required for HSC maintenance act in a short-range manner, which imposes the necessity for directional and positional cues in order for HSCs to localize and be retained properly in stem cell niches. The chemokine CXCL12 and its Gαi protein coupled receptor CXCR4, besides promoting HSC quiescence directly, also play instrumental roles in enabling HSCs to access bone marrow stem cell niches. Recent studies have revealed, however, that HSC niches also provide a constellation of hematopoietic cytokines that are critical for the production of most, if not all, blood cell types. Some hematopoietic cytokines, namely IL-7 and IL-15 produced by HSC niches, are not only required for lymphopoiesis but are also essential for memory T cell maintenance. Consequently, hematopoietic progenitors and differentiated immune cells, such as memory T cell subsets, also depend on the CXCL12/CXCR4 axis for migration into bone marrow and interactions with MSPCs and ECs. Similarly, subsets of antibody-secreting plasma cells also reside in close association with CXCL12-producing MSPCs in the bone marrow and require the CXCR4/CXCL12 axis for survival and long-term maintenance. Collectively, these studies demonstrate a broad range of key physiological roles, spanning blood cell production and maintenance of immunological memory, that are orchestrated by stem cell niches through a common and simple mechanism: CXCL12/CXCR4-mediated cell recruitment followed by receipt of a maintenance and/or instructive signal. A fundamental flaw of this type of cellular organization is revealed by myeloid and lymphoid leukemias, which target stem cell niches and induce profound transcriptomic changes that result in reduced hematopoietic activity and altered mesenchymal cell differentiation.

Acute myeloid leukemia–induced remodeling of the human bone marrow niche predicts clinical outcome

AbstractMurine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However, little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here, we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly, their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact–dependent manner through inhibition of cytoplasmic β-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30% lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore, patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7% whereas patients with higher osteocalcin levels reached a survival rate of 66.8%. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients.

Cobalt Chromium Molybdenum Surface Modifications Alter the Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells.

Surface roughness on orthopedic implant materials has been shown to be highly influential on the behavior of osteogenic cells. Mesenchymal stem and progenitor cells (MSPCs) migrate to the interface, adhere, proliferate, and differentiate into osteoblasts, which subsequently form bone matrix. Modifications of the implant surfaces should accelerate this process and improve biocompatibility. In this study, five surface topographies on cobalt chromium molybdenum (CoCrMo) were engineered to examine the influence on MSPCs. Scanning electron microscopy revealed significant differences in the morphology of untreated CoCrMo discs in comparison with CoCrMo with a titanium nitride (TiN) coating, polished and porous coated CoCrMo surfaces, and CoCrMo with a pure titanium (cpTi) coating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDX). Human primary MSPCs were expanded from tissue samples of spongiosa bone and characterized according to the criteria of the International Society for Cellular Therapy. The characteristic phenotype of MSPC was confirmed by flow cytometry and multilineage differentiation. Alcaline phosphatase and osteopontin expression increased significantly in all groups about 5-fold and 10-fold, respectively, in comparison to the undifferentiated controls. The porous coated surface showed a reduced expression of osteogenic markers. Due to the osteogenic differentiation, the expression of integrin α5β1, which is particularly important for cell-material contact, increased 4–7-fold. In the dynamic process of bone biology, MSPCs cultured and differentiated on cpTi, showed significant upregulation of IL6 and leptin.

Snai2 Maintains Bone Marrow Niche Cells by Repressing Osteopontin Expression

Bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs) are a critical constituent of the hematopoietic stem cell (HSC) niche. Previous studies have suggested that the zinc-finger epithelial-mesenchymal transition transcription factor Snai2 (also known as Slug) regulated HSCs autonomously. Here, we show that Snai2 expression in the BM is restricted to the BM stromal compartment where it regulates the HSC niche. Germline or MSPC-selective Snai2 deletion reduces the functional MSPC pool and their mesenchymal lineage output and impairs HSC niche function during homeostasis and after stress. RNA sequencing analysis revealed that Spp1 (osteopontin) expression is markedly upregulated in Snai2-deficient MSPCs. Genetic deletion of Spp1 in Snai2-deficient mice rescues MSPCs' functions. Thus, SNAI2 is a critical regulator of the transcriptional network maintaining MSPCs by the suppression of osteopontin expression.

Parathyroid Hormone Shifts Cell Fate of a Leptin Receptor-Marked Stromal Population from Adipogenic to Osteoblastic Lineage.

Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+ MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+ MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+ MSPC-derived adipogenesis in BM and increased LepR+ MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+ MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Low-dose radiation delays aging of human stem cells

Background & Aim Most tissues in the body harbour adult tissue stem cells (SCs) – multipotent, quiescent cells, capable of self-renewal, proliferation and differentiation into mature cellular subtypes. With the discovery of the unique properties of stem cells and their regenerative capacity came the possibility of utilizing these cells for therapeutic purposes. While very promising, stem cell therapy is thwarted by the challenge of obtaining donor-derived SCs in numbers that are sufficient to support therapy development with in vitro expansion leading to premature aging, loss of stemness and a significantly decreased functional and regenerative capacity of these cells. To address this challenge and based on the scientific reports of delayed aging following exposures to low-dose ionizing radiation in vitro and in vivo, we decided to investigate the effects of low-dose radiation (LDR) on cord-blood derived mesenchymal stem and progenitor cells (MSC/MSPC) and endothelial colony forming cells (ECFC). Methods, Results & Conclusion In our study, young, passage 4 stem cells were acutely irradiated with 10, 50 and 100 mGy gamma rays. Both SC types were aged in culture and their proliferation was monitored using Incucyte. Aging was defined as a decline in cell proliferative capacity and function. MSC function was evaluated based on the extent of chondrogenic differentiation. ECFC function was assessed by migration capacity in a scratch wound assay. The results of this study demonstrated that in vitro cultured non-irradiated MSCs and ECFCs aged significantly with cell doubling time increasing ∼3.0 fold. However, all doses of LDR treatment at early passage provided lasting effects and delayed aging, represented by improved proliferative capacity of 1.5-2.2 fold. Similarly, chondrogenic differentiation of aged MSCs decreased ∼2 fold but was restored back to young cell control levels with 50 and 100 mGy treatment. Interestingly, a 10 mGy exposure led to 50% further improvement exceeding the levels for young controls. The migratory capacity of aged ECFCs decreased ∼2 fold in comparison to young cells and was improved by ∼30% with LDR exposure. Thus, this report provides the first evidence of delayed aging and improved functional capacity of in vitro expanded mesenchymal and endothelial stem cells.

Acute Myeloid Leukemia Induced Remodelling of the Human Bone Marrow Niche Predicts Clinical Outcome

The hematopoietic stem cell (HSC) niche consists of different cellular and non-cellular constituents which regulate HSC maintenance and retention in the bone marrow. It has been shown in a number of murine models of myeloid neoplasia how leukemia infiltration alters the HSC niche to reinforce malignancy. Acute myeloid leukemia (AML) is characterized in human by a high relapse rate indicating that leukemia initiating cells are protected by its niche. However, despite our knowledge in murine models little is known about the bone marrow architecture in human and the impact of the leukemic niche on clinical outcome.In this study, we combined immunohistochemical stainings (IHC) with protein and global gene expression analyses together with clinical data to dissect the human bone marrow architecture in AML and assess its clinical impact. Human bone marrow was collected from AML patients at first diagnosis and matching non-leukemic donors. To evaluate the bone marrow architecture CD271+ mesenchymal stem and progenitor cells (MSPCs) were automatically quantified on bone marrow sections. In fact, AML patients showed 1.5-fold increase in bone marrow MSPCs compared to non-leukemic donors (Median (IQR), AML: 5.5% (2.8-9.5), n=36; control: 3.7% (2.1-5.7), n=58; p < 0.01). MSPCs proved to produce reticular fibers, an extracellular matrix protein frequently associated with different malignancies. In AML bone marrow these fibers were also found to be more abundantly expressed (Median (IQR), AML: 3.4% (1.8-4.5), n=37; control: 1.6% (1.1-3.3), n=19; p < 0.05).Next, to globally assess the gene expression profile of MSPCs in AML bone marrow we performed microarray analyses (ClariomTM S Human Assay) of freshly isolated uncultured lineage- CD146+ CD271+ MSPCs. Strikingly, HSC-regulating genes in particular CXCL12, ANGPT1 and VCAM1 showed lower expression in AML MSPCs which correlated with the degree of hematopoietic failure in AML patients. Along with the increased number of MSPCs, geneset enrichment analysis (GSEA) revealed higher proliferation of MSPCs in AML. In murine models loss of quiescence of MSPCs was previously found to be due to bone marrow sympathetic neuropathy. We therefore measured catecholamines and neurotrophic factor in the bone marrow extracellular fluid of AML patients and non-leukemic donors at first diagnosis. In fact, noradrenalin and brain-derived neurotrophic factor (BDNF) showed a 2-fold (p=0.26) resp. 4-fold (p<0.0001) lower expression in AML bone marrow. Importantly, BDNF is proved to be essential for sympathetic neuron proliferation and differentiation.In order to get an overview of alterations of canonical pathways in bone marrow MSPCs upon AML infiltration, we applied QIAGEN's Ingenuity® Pathway Analysis software. Several of the major differently regulated pathways proved to involve differentiation and mineralization of MSPCs. We therefore assessed bone metabolism in AML patients at first diagnosis and quantified serum osteocalcin levels. Notably, AML patients showed 30% lower osteocalcin levels than non-leukemic donors (Median (IQR), AML, 12.15ng/ml (7.53-16.28) n=58; control, 17.2ng/ml (12.5-23.45) n=31; p < 0.05). To evaluate if the deficiency in osteoblast mineralization is specifically due to AML infiltration we performed in vitro co-culture assays. Both MSPCs and an osteoblast-like cell line (SaOS2) showed significant impaired mineralization in presence of certain AML cell lines as well as primary human AML cells, while healthy mononuclear cells did not affect mineralization. Strikingly, this AML-induced defect in osteoblast mineralization proved to be of clinical significance. Patients with low osteocalcin levels (<11ng/ml) showed inferior overall survival with 1-year survival rate of 38.7% while patients with high osteocalcin levels reached 66.8% (n=58; median duration of follow-up 9.7 months).In summary, we globally characterized the bone marrow architecture in AML patients in comparison to non-leukemic donors and assessed its clinical significance. This increasing understanding of the human AML bone marrow microenvironment might open the window for new niche-targeted therapies to eradicate leukemic stem cells and eventually decrease the high relapse rate in AML. DisclosuresDuehrsen:Amgen: Research Funding; Roche: Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Honoraria.

3025 - Ebf1 as a Transcriptional Regulator of Bone Marrow Mesenchymal Stromal Cells

Mesenchymal stem and progenitor cells (MSPC), a crucial component of BM niche orchestrate maintenance, trafficking and stage specific differentiation of hematopoietic stem and progenitor cells (HSPCs). Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis, little is known about the transcriptional networks governing MSPCs biology. Here, we identified Early B-cell factor 1 (Ebf1) as new transcription regulator of MSPCs activity. MSPCs isolated from Ebf1-/- mice show diminished capacity to form fibroblastic colonies (CFU-F) indicating reduced self-renewal. Cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. Adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 in MSPCs in vivo leads to reduced numbers of HSPCs and myeloid cells in the bone marrow. Single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1 fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. HSPCs exposed to Ebf1-deficient niche exhibit reduced chromatin accessibility for AP-1, ETS, Runx and IRF transcription factors that are crucial for myeloid differentiation, which is consistent with decreased myeloid output observed in Prx1CreEbf1 fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. In conclusion, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.

Distinct roles of mesenchymal stem and progenitor cells during the development of acute myeloid leukemia in mice

AbstractDespite increasing evidence for the involvement of bone marrow (BM) hematopoietic stem cell niche in leukemogenesis, how BM mesenchymal stem and progenitor cells (MSPCs) contribute to leukemia niche formation and progression remains unclear. Using an MLL-AF9 acute myeloid leukemia (AML) mouse model, we demonstrate dynamic alterations of BM cellular niche components, including MSPCs and endothelial cells during AML development and its association with AML engraftment. Primary patient AML cells also induced similar niche alterations in xenografted mice. AML cell infiltration in BM causes an expansion of early B-cell factor 2+ (Ebf2+) MSPCs with reduced Cxcl12 expression and enhanced generation of more differentiated mesenchymal progenitor cells. Importantly, in vivo fate-mapping indicates that Ebf2+ MSPCs participated in AML niche formation. Ebf2+ cell deletion accelerated the AML development. These data suggest that native BM MSPCs may suppress AML. However, they can be remodeled by AML cells to form leukemic niche that might contribute to AML progression. AML induced dysregulation of hematopoietic niche factors like Angptl1, Cxcl12, Kitl, Il6, Nov, and Spp1 in AML BM MSPCs, which was associated with AML engraftment and partially appeared before the massive expansion of AML cells, indicating the possible involvement of the niche factors in AML progression. Our study demonstrates distinct dynamic features and roles of BM MSPCs during AML development.

Proliferation, migration and differentiation potential of human mesenchymal progenitor cells derived from osteoarthritic subchondral cancellous bone

Background: For regenerative therapies in the orthopedic field, one prerequisite for therapeutic success in the treatment of cartilage defects is the potential of body's own cells to migrate, proliferate and differentiate into functional cells. While this has been demonstrated for mesenchymal stem and progenitor cells (MPC) from healthy tissue sources, the potential of cells from degenerative conditions is unclear. In this study the regenerative potential of MPC derived from subchondral cancellous bone with diagnosed osteoarthritis is evaluated in vitro. Methods: OaMPC isolated from bone chips of three individual patients with Kellgren grade 3 osteoarthritis were characterized by analysis of cell surface antigen pattern. Cell proliferation was evaluated by doubling time and population doubling rate. Cell migration was assessed using a multi-well migration assay. Multi-lineage potential was evaluated by histological staining of adipogenic, osteogenic and chondrogenic markers. In addition, chondrogenic differentiation was verified by qPCR. Results: OaMPC showed a stable proliferation and a typical surface antigen pattern known from mesenchymal stem cells. Cell migration of oaMPC can be induced by human blood serum. OaMPC were capable of adipogenic, osteogenic and chondrogenic differentiation comparable to MPC derived from healthy conditions. Conclusion: OaMPC derived from knee joints affected by osteoarthritic conditions showed regeneration potential regarding migration, proliferation and chondrogenic differentiation. This suggests that oaMPC are able to contribute to cartilage repair tissue formation.

Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization during Bone Repair.

Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.

Chondrosarcoma: A Rare Misfortune in Aging Human Cartilage? The Role of Stem and Progenitor Cells in Proliferation, Malignant Degeneration and Therapeutic Resistance.

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is not clearly defined. However, it seems that mesenchymal stem and progenitor cells (MSPC) in the bone marrow facing a pro-proliferative as well as predominantly chondrogenic differentiation milieu, as is implicated in early stage osteoarthritis (OA) at that age, are the source of chondrosarcoma genesis. But how can MSPC become malignant? Indeed, only one person in 1,000,000 will develop a chondrosarcoma, whereas the incidence of OA is a thousandfold higher. This means a rare coincidence of factors allowing escape from senescence and apoptosis together with induction of angiogenesis and migration is needed to generate a chondrosarcoma. At early stages, chondrosarcomas are still assumed to be an intermediate type of tumor which rarely metastasizes. Unfortunately, advanced stages show a pronounced resistance both against chemo- and radiation-therapy and frequently metastasize. In this review, we elucidate signaling pathways involved in the genesis and therapeutic resistance of chondrosarcomas with a focus on MSPC compared to signaling in articular cartilage (AC).

EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis.Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. DisclosuresNo relevant conflicts of interest to declare.

Functional Inhibition of Mesenchymal Stem and Progenitor Cells (MSPC) Significantly Contributes to Hematopoietic Insufficiency with Acute Myeloid Leukemia (AML)

Pancytopenia is the most prominent clinical finding in patients with acute myeloid leukemia (AML) and represents a major cause for morbidity and mortality. So far, the underlying mechanisms leading to hematopoietic insufficiency in AML are poorly understood and therefore often mechanistically summarized as marrow replacement by infiltrating leukemic cells. Mesenchymal stem and progenitor cells (MSPC) are integral components of the bone marrow (BM) microenvironment and play an indispensable role for the regulation of normal hematopoiesis. Two AML mouse models have recently shown, that expansion of the leukemic clone leads to numeric changes and functional disturbances of niche components such as MSPC and osteoblasts, resulting in insufficient hematopoietic support (Hanoun et al. 2014; Frisch et al. 2012). As the knowledge about MSPC in human AML is limited so far, we conducted a detailed analysis of AML-derived MSPC in order to elucidate their contribution to hematopoietic failure.For this purpose we investigated the molecular and functional properties of BM-derived MSPC of 46 patients with AML covering all relevant subtypes according to WHO classification at diagnosis and/or during course of disease and compared them with MSPC functions of healthy controls. Hematopoietic insufficiency in the 31 patients at diagnosis was mirrored by 76% of the patients having bi- or pancytopenia and by median ANC of 1183/μl, median hemoglobin of 9.0 g/dl, and median platelet count of 67.000/μl. MSPC of these newly diagnosed patients exhibited significantly impaired growth capacities as shown by an altered morphology, reduced CFU-F activity, a lower number of passages and cumulative population doublings. While adipogenic differentiation potential was not affected, osteogenic differentiation potential of AML-derived MSPC was significantly reduced as indicated by cytochemical stainings, reduced Osterix and Osteocalcin (OC) mRNA levels as well as OC serum levels. Furthermore we detected altered mRNA and/or protein expression of key molecules involved in the regulation of hematopoietic stem and progenitor cells (HSPC), namely SCF, Angiopoietin-1, Jagged1 and Osteopontin. Functionally, this translated into a significantly diminished ability of AML-derived MSPC to support healthy CD34+ HSPC in LTC-IC assays.This insufficient stromal support was reversible and correlated with disease status, as LTC-IC frequency returned to normal values in patients in remission, but remained low in patients with refractory disease. Along with this we also observed a significant increase of OC serum levels in patients, who achieved complete remission. These data clearly suggests a direct causal relationship between the presence of leukemic cells and MSPC functionality. In further support of this idea we observed reduced proliferation and osteogenic differentiation of healthy MSPC following cultivation in conditioned media (CM) of 4 AML cell lines (THP-1, HL-60, MV4-11, MOLM-13). A comparable inhibitory effect of AML cells on healthy MSPC growth was seen in transwell-assays arguing in favor for a cell-contact independent mechanism.In summary, our data show that AML-derived MSPC are structurally and functionally altered resulting in an insufficient stromal support for normal hematopoiesis in AML. The correlation between clinical remission status and stromal support function together with the finding, that healthy MSC can adopt an AML-like phenotype when exposed to AML-CM suggest an instructive role of the leukemic precursor cells. DisclosuresDührsen:Celgene: Honoraria, Research Funding. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.

Myelodysplasia is in the niche: novel concepts and emerging therapies.

Myelodysplastic syndromes (MDSs) represent clonal disorders mainly of the elderly that are characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. The pathogenesis of MDS is thought to evolve from accumulation and selection of specific genetic or epigenetic events. Emerging evidence indicates that MDS is not solely a hematopoietic disease but rather affects the entire bone marrow microenvironment, including bone metabolism. Many of these cells, in particular mesenchymal stem and progenitor cells (MSPCs) and osteoblasts, express a number of adhesion molecules and secreted factors that regulate blood regeneration throughout life by contributing to hematopoietic stem and progenitor cell (HSPC) maintenance, self-renewal and differentiation. Several endocrine factors, such as erythropoietin, parathyroid hormone and estrogens, as well as deranged iron metabolism modulate these processes. Thus, interactions between MSPC and HSPC contribute to the pathogenesis of MDS and associated pathologies. A detailed understanding of these mechanisms may help to define novel targets for diagnosis and possibly therapy. In this review, we will discuss the scientific rationale of ‘osteohematology' as an emerging research field in MDS and outline clinical implications.

Acute Myelogenous Leukemia-Induced Sympathetic Neuropathy Promotes Malignancy in an Altered Hematopoietic Stem Cell Niche

Perivascular mesenchymal stem and progenitor cells (MSPCs) are critical for forming a healthy hematopoietic stem cell (HSC) niche. However, the interactions and influence of acute myelogenous leukemia (AML) stem cells with the microenvironment remain largely unexplored. We have unexpectedly found that neuropathy of the sympathetic nervous system (SNS) promotes leukemic bone marrow infiltration in an MLL-AF9 AML model. Development of AML disrupts SNS nerves and the quiescence of Nestin(+) niche cells, leading to an expansion of phenotypic MSPCs primed for osteoblastic differentiation at the expense of HSC-maintaining NG2(+) periarteriolar niche cells. Adrenergic signaling promoting leukemogenesis is transduced by the β2, but not β3, adrenergic receptor expressed on stromal cells of leukemic bone marrow. These results indicate that sympathetic neuropathy may represent a mechanism for the malignancy in order to co-opt the microenvironment and suggest separate mesenchymal niche activities for malignant and healthy hematopoietic stem cells in the bone marrow.