Mesenchymal stem cells in hematopoietic stem cell transplantation

Mesenchymal stromal/stem cells (MSC) of bone marrow (BM) origin not only provide the supportive microenvironmental niche for hematopoietic stem cells (HSC) but are capable of differentiating into various cell types of mesenchymal origin, such as bone, fat and cartilage. In vitro and in vivo data suggest that MSC have low inherent immunogenicity, modulate/suppress immunologic responses through interactions with immune cells, and home to damaged tissues to participate in regeneration processes through their diverse biologic properties. MSC derived from BM are being evaluated for a wide range of clinical applications, including disorders as diverse as myocardial infarction and newly diagnosed diabetes mellitus type 1. However, their use in HSC transplantation, either for enhancement of hematopoietic engraftment or for treatment/prevention of graft-versus-host disease, is far ahead of other indications. Ease of isolation and ex vivo expansion of MSC, combined with their intriguing immunomodulatory properties and their impressive record of safety in a wide variety of clinical trials, make these cells promising candidates for further investigation.     

Biochemical changes induced by strontium ranelate in differentiating adipocytes

Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1–2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu.     

Preconditioning influences mesenchymal stem cell properties in vitro and in vivo

Various diseases and toxic factors easily impair cellular and organic functions in mammals. Organ transplantation is used to rescue organ function, but is limited by scarce resources. Mesenchymal stem cell (MSC)‐based therapy carries promising potential in regenerative medicine because of the self‐renewal and multilineage potency of MSCs; however, MSCs may lose biological functions after isolation and cultivation for a long time in vitro. Moreover, after they are injected in vivo and migrate into the damaged tissues or organs, they encounter a harsh environment coupled with death signals due to the inadequate tensegrity structure between the cells and matrix. Preconditioning, genetic modification and optimization of MSC culture conditions are key strategies to improve MSC functions in vitro and in vivo, and all of these procedures will contribute to improving MSC transplantation efficacy in tissue engineering and regenerative medicine. Preconditioning with various physical, chemical and biological factors is possible to preserve the stemness of MSCs for further application in studies and clinical tests. In this review, we mainly focus on preconditioning and the corresponding mechanisms for improving MSC activities in vitro and in vivo; we provide a glimpse into the promotion of MSC‐based cell therapy development for regenerative medicine. As a promising consequence, MSC transplantation can be applied for the treatment of some terminal diseases and can prolong the survival time of patients in the near future.     

Subcutaneous graft of D1 mouse mesenchymal stem cells leads to the formation of a bone-like structure

Mesenchymal stem cells (MSC) are capable of both self-renewal and multi-lineage differentiation into mesoderm-type cells such as osteoblasts, chondrocytes, adipocytes and myocytes. Together the multipotent nature of MSCs and the facility to expand them in vitro make these cells ideal resources for regenerative medicine, particularly for bone reconstruction, and therefore research efforts focused on defining efficient protocols for directing their differentiation into the requisite lineage. Despite much progress in identifying mechanisms and factors that direct and control in vitro osteogenic differentiation of MSCs, a rapid and simple model to evaluate in vivo tissue formation is still lacking. Here, we describe the unique capacity of the murine bone marrow-derived D1 MSC cell line, which differentiates in vitro into at least three cell lineages, to form in vivo a structure resembling bone. This bone-like structure was obtained after subcutaneous grafting of D1 cells into immunocompetent mice without the need of neither an osteogenic factor nor scaffold material. These data allow us to propose this cell model as a tool for exploring in vivo the mechanisms and/or factors that govern and potentially regulate osteogenesis.     

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy

Background aimsTo obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo.MethodsA retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo.ResultsThe cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages).ConclusionsThese results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs.     

Artificial intelligence for compound pharmacokinetics prediction

Optimisation of compound pharmacokinetics (PK) is an integral part of drug discovery and development. Animal in vivo PK data as well as human and animal in vitro systems are routinely utilised to evaluate PK in humans. In recent years machine learning and artificial intelligence (AI) emerged as a major tool for modelling of in vivo animal and human PK, enabling prediction from chemical structure early in drug discovery, and therefore offering opportunities to guide the design and prioritisation of molecules based on relevant in vivo properties and, ultimately, predicting human PK at the point of design. This review presents recent advances in machine learning and AI models for in vivo animal and human PK for small-molecule compounds as well as some examples for antibody therapeutics.     

Featured Article: Dexamethasone and rosiglitazone are sufficient and necessary for producing functional adipocytes from mesenchymal stem cells

The final product of adipogenesis is a functional adipocyte. This mature cell acquires the necessary machinery for lipid metabolism, loses its proliferation potential, increases its insulin sensitivity, and secretes adipokines. Multipotent mesechymal stromal cells have been recognized as a source of adipocytes both in vivo and in vitro. The in vitro adipogenic differentiation of human MSC (hMSC) has been induced up to now by using a complex stimulus which includes dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin (a classical cocktail) and evaluated according to morphological changes. The present work was aimed at demonstrating that the simultaneous activation of dexamethasone’s canonical signaling pathways, through the glucocorticoid receptor and CCAAT-enhancer-binding proteins (C/EBPs) and rosiglitazone through peroxisome proliferator-activated receptor gamma (PPAR-gamma) is sufficient yet necessary for inducing hMSC adipogenic differentiation. It was also ascertained that hMSC exposed just to dexamethasone and rosiglitazone (D&R) differentiated into cells which accumulated neutral lipid droplets, expressed C/EBP-alpha, PPAR-gamma, aP2, lipoprotein lipase, acyl-CoA synthetase, phosphoenolpyruvate carboxykinase, adiponectin, and leptin genes but did not proliferate. Glucose uptake was dose dependent on insulin stimulus and high levels of adipokines were secreted (i.e. displaying not only the morphology but also expressing mature adipocytes’ specific genes and functional characteristics). This work has demonstrated that (i) the activating C/EBPs and PPAR-gamma signaling pathways were sufficient to induce adipogenic differentiation from hMSC, (ii) D&R producing functional adipocytes from hMSC, (iii) D&R induce adipogenic differentiation from mammalian MSC (including those which are refractory to classical adipogenic differentiation stimuli). D&R would thus seem to be a useful tool for MSC characterization, studying adipogenesis pathways and producing functional adipocytes.     

Mesenchymal stromal/stem cells markers in the human bone marrow

Background aimsMesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation.MethodsTo characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression.ResultsMicroscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers.ConclusionsTargeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.     

Local Delivery of Mesenchymal Stem Cells with Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold Suppress Arthritis in Rats

Mesenchymal stem cells (MSC) have been used recently for the treatment of autoimmune diseases in murine animal models due to the immunoregulatory capacity. Current utilization of MSC requires cells in certain quantity with multiple courses of administration, leading to limitation in clinical usage. Here we efficiently treated collagen-induced arthritis rats with a single local implantation with reduced number of MSC (2∼20% of previous studies) with nano-fiber poly-lactic-co-glycolic acid (nano-fiber) scaffold. MSC seeded on nano-fiber scaffold suppressed arthritis and bone destruction due to inhibition of systemic inflammatory reaction and immune response by suppressing T cell proliferation and reducing anti- type II collagen antibody production. In vivo tracing of MSC demonstrated that these cells remained within the scaffold without migrating to other organs. Meanwhile, in vitro culture of MSC with nano-fiber scaffold significantly increased TGF-β1 production. These results indicate an efficient utilization of MSC with the scaffold for destructive joints in rheumatoid arthritis by a single and local inoculation. Thus, our data may serve as a new strategy for MSC-based therapy in inflammatory diseases and an alternative delivery method for bone destruction treatment.     

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects

Background aimsCombining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.MethodsMSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.ResultsEffective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.ConclusionsMSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.     

A sensitive in vitro assay for the detection of residual viable rabies virus in inactivated rabies vaccines

Rabies is a viral disease transmitted through bites from rabid animals and can be prevented by vaccines. Clinically used rabies vaccines are prepared from inactivated rabies viruses grown in cell cultures or embryonated eggs. In Japan and across the world, tests that confirm complete inactivation, such as the in vivo suckling mouse assay, in which suckling mice are intracerebrally inoculated with vaccine products, are required for quality control. In this study, we developed a novel cell-based immunofluorescence assay that does not require mice for testing rabies vaccine inactivation for human use. The sensitivity of this cell-based in vitro assay was 5.7 times that of the in vivo suckling mouse assay, with a detection limit of one focus forming units per ml of test sample. This newly developed in vitro assay may replace the established in vivo suckling mouse assay for confirming viral vaccine inactivation.     

Proscillaridin A induces apoptosis and inhibits the metastasis of osteosarcoma in vitro and in vivo

The side effects of chemotherapy, drug resistance, and tumor metastasis hinder the development of treatment for osteosarcoma, leading to poor prognosis of patients with the disease. Proscillaridin A, a kind of cardiac glycoside, has been proven to have anti-proliferative properties in many malignant tumors, but the efficacy of the drug in treating osteosarcoma is unclear. In the present study, we assessed the effects of Proscillaridin A on osteosarcoma and investigated its underlying action mechanism. The cell cytotoxicity assay showed that Proscillaridin A significantly inhibited the proliferation of 143B cells in a dose- and time-dependent manner. Also, flow cytometry and invasion assay revealed that Proscillaridin A induced apoptosis and reduced 143B cell motility. Western blotting and PCR were used to detect the expressions of Bcl-xl and MMP2 and showed that mRNA/protein expression levels decreased significantly in Proscillaridin A-treated osteosarcoma cells. Using a mouse xenograft model, we found that Proscillaridin A treatment significantly inhibited tumor growth and lung metastasis in vivo and decreased the expression levels of Bcl-xl and MMP2. No noticeable side effect was observed in the liver, kidney, and hematological functions. Conclusively, Proscillaridin A suppressed proliferation, induced apoptosis, and inhibited 143B cell metastasis in vitro and in vivo, and these effects could be mediated by downregulating the expressions of Bcl-xl and MMP2.     

Geranylgeranyl transferase type II inhibition prevents myeloma bone disease

Geranylgeranyl transferase II (GGTase II) is an enzyme that plays a key role in the isoprenylation of proteins. 3-PEHPC, a novel GGTase II inhibitor, blocks bone resorption and induces myeloma cell apoptosis in vitro. Its effect on bone resorption and tumor growth in vivo is unknown. We investigated the effect of 3-PEHPC on tumor burden and bone disease in the 5T2MM model of multiple myeloma in vivo. 3-PEHPC significantly reduced osteoclast numbers and osteoclast surface. 3-PEHPC prevented the bone loss and the development of osteolytic bone lesions induced by 5T2MM myeloma cells. Treatment with 3-PEHPC also significantly reduced myeloma burden in bone. The magnitude of response was similar to that seen with the bisphosphonate, risedronate. These data show that targeting GGTase II with 3-PEHPC can prevent osteolytic bone disease and reduce tumor burden in vivo, and represents a novel approach to treating tumors that grow in bone.     

Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only…

The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.     

The role of mesenchymal stromal cells in spinal cord injury,regenerative medicine and possible clinical applications

Diseases of the central nervous system still remain among the most challenging pathologies known to mankind, having no or limited therapeutic possibilities and a very pessimistic prognosis. Advances in stem cell biology in the last decade have shown that stem cells might provide an inexhaustible source of neurons and glia as well as exerting a neuroprotective effect on the host tissue, thus opening new horizons for tissue engineering and regenerative medicine. Here, we discuss the progress made in the cell-based therapy of spinal cord injury. An emphasis has been placed on the application of adult mesenchymal stromal cells (MSCs). We then review the latest and most significant results from in vitro and in vivo research focusing on the regenerative/neuroprotective properties of MSCs. We also attempt to correlate the effect of MSCs with the pathological events that are taking place in the nervous tissue after SCI. Finally, we discuss the results from preclinical and clinical trials involving different routes of MSC application into patients with neurological disorders of the spinal cord.     

The B subunit of Escherichia coli heat‐labile toxin alters the development and antigen‐presenting capacity of dendritic cells

Escherichia coli's heat‐labile enterotoxin (Etx) and its non‐toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co‐administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC‐II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c⁺ DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen‐presenting cells on encounter with T cells.     

Collective behaviours in organoids

Collective behaviour emerges from interacting units within communities, such as migrating herds, swimming fish schools, and cells within tissues. At the microscopic level, collective behaviours include collective cell migration in development and cancer invasion, rhythmic gene expression in pattern formation, cell competition in homeostasis and cancer, force generation and mechano-sensing in morphogenesis. Studying the initiation and the maintenance of collective cell behaviours is key to understand the principles of development, regeneration and disease. However, the manifold influences of contributing factors in in vivo environments challenge the dissection of causalities in animal models. As an alternative model that has emerged to overcome this difficulty, in vitro three-dimensional organoid cultures provide a reductionist approach yet retain similarities with the in vivo tissue in cellular composition and tissue organisation. Here, we focus on recent progresses in studying collective behaviours in different organoid systems and discuss their advantages and the possibility of improvement for future applications.     

Mesenchymal stem cell aging: Mechanisms and influences on skeletal and non-skeletal tissues

The aging population and the incidence of aging-related diseases such as osteoporosis are on the rise. Aging at the tissue and organ levels usually involves tissue stem cells. Human and animal model studies indicate that aging affects two aspects of mesenchymal stem cell (MSC): a decrease in the bone marrow MSC pool and biased differentiation into adipocyte at the cost of osteoblast, which underlie the etiology of osteoporosis. Aging of MSC cells is also detrimental to some non-skeletal tissues, in particular the hematopoietic system, where MSCs serve as a niche component. In addition, aging compromises the therapeutic potentials of MSC cells, including cells isolated from aged individuals or cells cultured for many passages. Here we discuss the recent progress on our understanding of MSC aging, with a focus on the effects of MSC aging on bone remodeling and hematopoiesis and the mechanisms of MSC aging.