Mesenchymal Stem/progenitor Cells Research Articles

Under Pressure: When a Transformed Environment Pushes Cells to Malignancy

Mesenchymal stem/progenitor cells (MSPCs) have recently gained attention as key elements that contribute to different stages of oncogenesis, from predisposition to disease manifestation and evolution. In this issue of Cell Stem Cell, Zambetti etal. (2016) propose a mechanism by which mutated MSPCs transform hematopoietic cells into a malignant-prone state.

Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration.

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified. CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed. The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential. Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.

Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration

Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF’s bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.

Engineering Human TMJ Discs with Protein-Releasing 3D-Printed Scaffolds

The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D)–printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) were then delivered in the scaffolds by spatially embedding CTGF- or TGFβ3-encapsulated microspheres (µS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TGFβ3-µS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TGFβ3-µS (100 mg µS/g of scaffold) showed significantly more collagen II and aggrecan in the intermediate zone than a low dose (50 mg µS/g of scaffold). Similarly, a high dose of CTGF/TGFβ3-µS yielded significantly higher collagen I expression in the AP bands compared with the low-dose and empty µS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TGFβ3-µS than empty µS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TGFβ3-µS compared with the low-dose and empty µS, suggesting the dose effect of CTGF and TGFβ3 on fibrocartilage formation. Together, our findings may represent an efficient approach to engineering the TMJ disc graft with anisotropic scaffold microstructure, heterogeneous fibrocartilaginous matrix, and region-dependent viscoelastic properties.

Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair in a mouse injury model.

Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ “super-healer” strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

Cell Autonomous Defects of Mesenchymal Stem Progenitor Cells of Patients with CMML Contribute to Impaired Hematopoietic Supportive Activity

Objective: Increasing evidence indicates a critical role of the hematopoietic microenvironment in the pathophysiology of myeloid malignancies. Mesenchymal stem/progenitor cells (MSPCs) represent an indispensible cellular component of the bone marrow microenvironment and play an important role in supporting and regulating the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). Abnormal biological characteristics of MSPCs involved in the initiating and development of hematopoietic disorders, such as chronic myelogenous leukemia and myelodysplastic syndromes. However, the characteristics and the role of primary MSPC defects in the pathogenesis of chronic myelomonocytic leukemia (CMML) remain poorly understood.Methods: MSPCs were generated from bone marrow aspirates of patients with CMML and that of healthy controls. To investigate the frequency of the MSPCs in the bone marrow of CMML patients and healthy controls in vivo, a colony-forming unit fibroblast (CFU-F) assay was performed. The phenotypic evaluation of the MSPCs was performed with flow cytometric analysis after staining with a cocktail of cell surface markers, including CD34, CD45, CD29, CD44, CD73 and CD105. To determine the functional difference between the CMML- and control-MSPCs, senescence, cell proliferation, and cell cycle were assessed by EDU incorporation assays, β-galactosidase activity, and flow cytometric analysis (following BrdU/7AAD staining, respectively. To determine the multi-lineage potential of MSPCs, MSPCs were subjected osteoblast, chondrocyte and adipocyte differentiation cultures. The hematopoietic supportive activity was assessed using cobblestone-area forming cell (CAFC) assays by coculturing of CD34+ cord blood cells with CMML- or control- MSPCs. Furthermore, to evaluate the soluble factors of these MSPCs in HSPC supportive activity, the transwell assay was performed with a subsequent flow cytometric analysis further determine the multi-lineage differentiation alteration. Cytokine array was used to determine the cytokines/growth factors in the MSPC conditioned media.Results: The expression levels of the surface markers were identical between CMML-MSPCs and control MSPCs. The frequency of CFU-F is identical between healthy control and CMML patients. However, CMML-MSPCs exhibited a poor proliferative capacity compared to controls MSPCs. CMML MSPCs exhibited an increased senescence and a S phase retardation in the cell cycle as assessed microscopically and flow cytometrically, respectively. Differentiation assay revealed that CMML MSPCs had increased chondrocyte differentiation and reduced adipocyte differentiation, while no alteration of osteoblast differentiation was noticed. In vitro co-cultures of MSPCs and CD34+ umbilical cord blood cells demonstrated that CMML-MSPCs had an impaired hematopoietic supportive capability as compared to control MSPCs as evidenced by reduced numbers of cobble-stone and reduced numbers of CFU-C. Furthermore, a reduced CFU-C and a reduced CFU-GEMM were observed in CAFC and transwell cultures containing CMML-MSPCs compared to that of control MSPCs. In contrast, an increased myeloid cells (CD33+/CD14-/CD15+) and an increased CFU-GM were observed in CD34+ cells and CMML-MSPC co-cultures as compared to that in CD34+ cells and control-MSPC co-cultures. Cytokine array showed that the conditional media of CMML-MSPCs has lower levels of multiple cytokines (IL-6, IL-8, and GRO-β) compared to that of control MSPC conditional media.Conclusion: Collectively, our study indicated that MSPCs of CMML patients had reduced proliferation, increased senescence and reduced cytokine production, while differentiation was not markedly altered. CMML-MSPCs display less hematopoietic supportive activity. Furthermore, CMML-MSPCs induced CD34+ cell differentiation favoring granulomonocytic lineage over erythroid cells. Our study suggests that the cell autonomous defects of CMML-MSPCs contribute to the pathogenesis of CMML and the interaction between hematopoietic compartment and microenvironment could serve as targets for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

A robust potency assay highlights significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radio-resistance.

The inherent immunomodulatory capacity of mesenchymal stem/progenitor cells (MSPCs) encouraged initiation of multiple clinical trials. Release criteria for therapeutic MSPCs cover identity, purity and safety but appropriate potency assessment is often missing. Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection. We established a robust immunomodulation potency assay based on pooling responder leukocytes to minimize individual immune response variability. Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis. Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0233-8) contains supplementary material, which is available to authorized users.

T15: Circulating tumor cells and bone marrow progenitor cells in the blood of breast cancer patients in the dynamics of neoadjuvant chemotherapy

Background Despite the abundance of theoretical evidences displaying the considerable role of the “soil” in metastasis progression, the majority of cancer research and cancer therapy pathogenetic methods focused mainly on the tumor cells. There is still no clear clinical data showing the role of “soil” in the metastasis. Identification of factors and mechanisms driving the conditions promoting tumor dissemination may lead to therapeutic strategy to detect and prevent metastases at the earliest step. It has been established that bone marrow-derived hematopoietic progenitor cells home to pre-metastatic sites before tumor cells infiltrate in these sites. These hematopoietic progenitor cells form regulatory cell cluster of stromal microenvironment (premetastatic niche) to promote secondary tumor growth. In this context, the aim of our study was to evaluate the level of different pools of circulating tumor cells and bone marrow progenitor cells in the blood of patients with breast cancer in the dynamics of neoadjuvant chemotherapy. Materials and methods Patients (prospective study) with newly diagnosed invasive breast cancer in the age range 18–50 years and tumor volume of 2.0(> or =) cm, referred at the Tomsk Cancer Research Institute for treatment were included into the study. Eligibility criteria were as follows: the patient’s informed consent to participate in research; morphologically verified diagnosis of invasive carcinoma of non-specific type; luminal B-1, -2, triple negative (basal-like subtype included), HER2 positive breast cancer; T2-4N0-3M0; preserved menstrual function; satisfactory performance status (on a scale ( Results The study showed that each succeeding course of NACHT increased blood levels of circulating tumor cells, and bone marrow progenitor cells with a phenotype characteristic of EPC (endothelial progenitor cells) (CD34 + CD45–VEGFR + CD133 + CD202 +) and MSC (mesenchymal progenitor stem cells fibroblasts) (CD34–CD90 + VEGFR1–CD45–CD202–). Influence of the NACHT on hematopoietic stem cells HSC (VEGFR1 + CD34 + CD45lowSD202–) and progenitor cells HPC – CD34 + CD45 + CD90–VEGFR1 + CD133 – was ambiguous. Conclusion Neoadjuvant chemotherapy increases blood levels of circulating tumor cells, endothelial progenitor cells and mesenchymal stem cells progenitor fibroblasts, thus promoting the formation of premetastatic niche. The study was conducted with financial support from the Council for Grants of the President of the Russian Federation (Russia) for the state support of young philosophy doctors, agreements of SC 114.120.14.168-MD Russian Scientific Foundation (Russia), Grant No. 14-15-00318 (sample collection) and Russian Foundation for Basic Research (Russia), Grant No. 15-34-20864 . We acknowledge support of this work by the Tomsk State University Competitiveness Improvement Program (Russia).

Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells From Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration.

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and antiapoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1+/−) mice. Nf1+/− MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1+/− MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1+/− MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

Transient gamma-secretase inhibition accelerates and enhances fracture repair likely via Notch signaling modulation

Approximately 10% of skeletal fractures result in healing complications and non-union, while most fractures repair with appropriate stabilization and without pharmacologic intervention. It is the latter injuries that cannot be underestimated as the expenses associated with their treatment and subsequent lost productivity are predicted to increase to over $74 billion by 2015. During fracture repair, local mesenchymal stem/progenitor cells (MSCs) differentiate to form new cartilage and bone, reminiscent of events during skeletal development. We previously demonstrated that permanent loss of gamma-secretase activity and Notch signaling accelerates bone and cartilage formation from MSC progenitors during skeletal development, leading to pathologic acquisition of bone and depletion of bone marrow derived MSCs. Here, we investigated whether transient and systemic gamma-secretase and Notch inhibition is capable of accelerating and enhancing fracture repair by promoting controlled MSC differentiation near the fracture site. Our radiographic, microCT, histological, cell and molecular analyses reveal that single and intermittent gamma-secretase inhibitor (GSI) treatments significantly enhance cartilage and bone callus formation via the promotion of MSC differentiation, resulting in only a moderate reduction of local MSCs. Biomechanical testing further demonstrates that GSI treated fractures exhibit superior strength earlier in the healing process, with single dose GSI treated fractures exhibiting bone strength approaching that of un-fractured tibiae. These data further establish that transient inhibition of gamma-secretase activity and Notch signaling temporarily increases osteoclastogenesis and accelerates bone remodeling, which coupled with the effects on MSCs likely explains the accelerated and enhanced fracture repair. Therefore, we propose that the Notch pathway serves as an important therapeutic target during skeletal fracture repair.

Are clinical trials with mesenchymal stem/progenitor cells too far ahead of the science? Lessons from experimental hematology.

The cells referred to as mesenchymal stem/progenitor cells (MSCs) are currently being used to treat thousands of patients with diseases of essentially all the organs and tissues of the body. Strikingly positive results have been reported in some patients, but there have been few prospective controlled studies. Also, the reasons for the beneficial effects are frequently unclear. As a result there has been a heated debate as to whether the clinical trials with these new cell therapies are too far ahead of the science. The debate is not easily resolved, but important insights are provided by the 60-year history that was required to develop the first successful stem cell therapy, the transplantation of hematopoietic stem cells. The history indicates that development of a dramatically new therapy usually requires patience and a constant dialogue between basic scientists and physicians carrying out carefully designed clinical trials. It also suggests that the field can be moved forward by establishing better records of how MSCs are prepared, by establishing a large supply of reference MSCs that can be used to validate assays and compare MSCs prepared in different laboratories, and by continuing efforts to establish in vivo assays for the efficacy of MSCs. Stem Cells 2014;32:3055–3061

Interleukin 8/KC enhances G-CSF induced hematopoietic stem/progenitor cell mobilization in Fancg deficient mice.

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by a progressive bone marrow aplasia, chromosomal instability, and acquisition of malignancies. Successful hematopoietic cell transplantation (HCT) for FA patients is challenging due to hypersensitivity to DNA alkylating agents and irradiation of FA patients. Early mobilization of autologous stem cells from the bone marrow has been thought to be ideal prior to the onset of bone marrow failure, which often occurs during childhood. However, the markedly decreased response of FA hematopoietic stem cells to granulocyte colony-stimulating factor (G-CSF) is circumventive of this autologous HCT approach. To-date, the mechanism for defective stem cell mobilization in G-CSF treated FA patients remains unclear. Fancg heterozygous (Fancg (+/-)) mice utilized in these studies. Student's t-test and one-way ANOVA were used to evaluate statistical differences between WT and Fancg (-/-) cells. Statistical significance was defined as P values less than 0.05. Fancg deficient (Fancg (-/-)) mesenchymal stem/progenitor cells (MSPCs) produce significant lower levels of KC, an interleukin-8 (IL-8) related chemoattractant protein in rodents, as compared to wild type cells. Combinatorial administration of KC and G-CSF significantly increased the mobilization of hematopoietic stem/progenitor cells (HSPCs) in Fancg (-/-) mice. In summary, our results suggest that KC/IL-8 could be proved useful in the synergistic mobilization of FA HSPCs in combination with G-CSF.

The role of mesenchymal stem/progenitor cells in sarcoma: update and dispute.

Sarcoma is the collective name for a relatively rare, yet heterogeneous group of cancers, most probably derived from mesenchymal tissues. There are currently over 50 sarcoma subtypes described underscoring the clinical and biologic diversity of this group of malignant cancers. This wide lineage range might suggest that sarcomas originate from either many committed different cell types or from a multipotent cell. Mesenchymal stem/progenitor cells (MSCs) are able to differentiate into many cell types and these multipotent cells have been isolated from several adult human tumors, making them available for research as well as potential beneficial therapeutical agents. Recent accomplishments in the field have broadened our knowledge of MSCs in relation to sarcoma origin and sarcoma treatment in therapeutic settings. However, numerous concerns and disputes have been raised about whether they are the putative originating cells of sarcoma and their questionable role in sarcomagenesis and progression. We summarize the update and dispute about MSC investigations in sarcomas including the definition, cell origin hypothesis, functional and descriptive assays, roles in sarcomagenesis and targeted therapy, with the purpose to give a comprehensive view of the role of MSCs in sarcomas.

Suppression of spinal cord injury on human mesenchymal stem/progenitor cells (MSCs) mediated by pituitary adenylate cyclase activating polypeptide (PACAP)

Suppression of spinal cord injury on human mesenchymal stem/progenitor cells (MSCs) mediated by pituitary adenylate cyclase activating polypeptide (PACAP)

Why Mesenchymal Stem/Progenitor Cell Heterogeneity in Specific Environments? <br/>—Implications for Tissue Engineering Applications Following Injury or Degeneration of Connective Tissues

Mesenchymal stem/progenitor cells (MSC/MPC) from a variety of tissue sources (bone marrow, adipose tissue, fat pads, synovial membranes, synovial fluid, skin, muscle and periosteal tissue) have been widely applied for tissue engineering applications to generate replacements for injured or degenerated tissues. Alternatively, they have also been injected as free cells in an attempt to facilitate in vivo repair. Nearly all studies reported have used mixed cell populations of MSC/MPC, usually defined by cell surface phenotypes and/or functional ability to differentiate towards multiple cell lineages. Using more detailed cell surface phenotyping and limiting dilution approaches to isolate individual MSC/MPC clones have indicated that such mixed cell populations are very heterogeneous. In addition subsets of cells from different sources may have epigenetic modifications. While it is clear that MSC/MPC cells exhibit heterogeneity, the question of why this is the case has not been well addressed. This review will address some of these issues, as well as provide some insights into the implications when using such diverse cells for tissue engineering applications.

Is Adipocyte Differentiation the Default Lineage for Mesenchymal Stem/Progenitor Cells after Loss of Mechanical Loading? A Perspective from Space Flight and Model Systems

Mesenchymal stem/progenitor cells (MSC/MPC) are found in many tissues and fluids including bone marrow, adipose tissues, muscle, synovial membranes, synovial fluid, and blood. Such cells from different sources can proliferate and differentiate into different lineages (e.g. osteogenic, chondrogenic and adipogenic) after suitable stimulation. However, details regarding the regulation of MSC/MPC proliferation and differentiation status are still unclear and it is likely that regulation involves both biological and mechanical influences in the different environments. It has been noted that in humans and preclinical animal models that exposure to microgravity/space flight or prolonged bed rest (a surrogate for microgravity) can lead to infiltration of skeletal muscle and bone marrow with fat. Similarly, in preclinical models treated with multiple intramuscular injections of Botulinum Toxin A to induce muscle weakness and atrophy, there is also an infiltration of the muscle with fat. The origins and basis for these fat deposits are largely unknown, but there is a possibility that the altered mechanical and biological environments lead to dysregulation of MSC/MPC and progression to preferential differentiation towards the adipocyte lineage. Furthermore, loss of MSC regulatory control by either mechanical and/or biological factors may also contribute to their involvement in obesity development and progression. Thus, the utility of using MSC/MPC from some sources for tissue engineering purposes may be compromised and further research regarding optimal loading for tissue engineering purposes is likely warranted.

Human and Murine β-Defensin-Derived Peptides Induce Rapid Mobilization Of Murine Hematopoietic Stem and Progenitor Cells Via Activation Of CXCR4 Signaling and CXCL12 Release

BackgroundRapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization. ResultsWhile AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation. ConclusionsOur study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation. Disclosures:Scadden:Fate Therapeutics: Consultancy, Equity Ownership.

Identification and Isolation of Small CD44-Negative Mesenchymal Stem/Progenitor Cells From Human Bone Marrow Using Elutriation and Polychromatic Flow Cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.