Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine

Mesenchymal stem cells (MSCs) are partially defined by their ability to differentiate into tissues including bone, cartilage and adipose in vitro, but it is their trophic, paracrine and immunomodulatory functions that may have the greatest therapeutic impact in vivo. Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues. Significant progress has been made in understanding the biochemical and metabolic mechanisms and feedback associated with MSC response. The anti-inflammatory and immunomodulatory capacity of MSC may be paramount in the restoration of localized or systemic conditions for normal healing and tissue regeneration. Allogeneic MSC treatments, categorized as a drug by regulatory agencies, have been widely pursued, but new studies demonstrate the efficacy of autologous MSC therapies, even for individuals affected by a disease state. Safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications.     

Intramarrow injection of β-catenin-activated,but not na?ve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with β-catenin-activated MSCs. These results reveal that naïve MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/β-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.     

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.     

Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.     

Change in serum proteome during allogeneic hematopoietic stem cell transplantation and clinical significance of serum C-reactive protein and haptoglobin

Successful hematopoietic stem cell transplantation (HSCT) involves the restoration of hematopoietic function after engraftment, arising from the differentiation and proliferation of hematopoietic stem cells. Several factors could influence the course of allogeneic-HSCT (allo-HSCT). Therefore, knowledge of serum proteome changes during the allo-HSCT period might increase the efficacy of diagnosis and disease prevention efforts. This study conducted proteomic analyses to find proteins that were significantly altered in response to allo-HSCT. Sera from five representative patients who underwent allo-HSCT were analyzed by 2-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, and were measured on a weekly basis before and after allo-HSCT in additional 78 patients. Fourteen protein spots showing changes in expression were further examined, and most proteins were identified as acute phase proteins (APPs). Studies of 78 additional patients confirmed that C-reactive protein (CRP) and haptoglobin undergo expression changes during allo-HSCT and thus may have the potential to serve as representative markers of clinical events after allo-HSCT. Maximal CRP level affected the development of major transplant-related complications (MTCs) and other problems such as fever of unknown origin. Particularly, an increase in CRP level 21 days after allo-HSCT was found to be an independent risk factor for MTC. Maximal haptoglobin and haptoglobin level 14 days after allo-HSCT were predictive of relapses in underlying hematologic disease. Our results indicated that CRP and haptoglobin were significantly expressed during allo-HSCT, and suggest that their level can be monitored after allo-HSCT to assess the risks of early transplant-related complications and relapse.     

Effect of F68 on Cryopreservation of Mesenchymal Stem Cells Derived from Human Tooth Germ

The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer's disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.     

A micro/nanoscale surface mechanical study on morpho-functional changes in multilineage-differentiated human mesenchymal stem cells

In recent years MSCs have become a very attractive tool in tissue engineering and regenerative medicine because of their ability to be committed along several lineages through chemical or physical stimuli. Nevertheless their therapeutic potential and plasticity are not yet totally understood. This report describes the use of AFM together with conventional microscopies to obtain mechanical information on cell surfaces and deposited extra cellular matrix molecules, after inducing the differentiation of human MSCs towards three typical mesoderm phenotypes. The aim is to correlate morphological, functional, and mechanical aspects of human MSCs to obtain a deeper understanding of their great potential.     

Microtopography based on inverse opal structures regulates the behavior of bone marrow‐derived mesenchymal stem cells

Physical cues from the extracellular microenvironment play an important role in regulating cell behavior, such as adhesion, migration, and differentiation. Many studies have shown that different physical parameters (eg, stiffness and topography) could modulate the in vitro differentiation of mesenchymal stem cells (MSCs), which had multilineage differentiation potential and could be easily isolated from various tissues such as bone marrow, adipose tissue, and the umbilical cord. However, the underlying mechanism of the topographical influence on MSCs and the detailed cell‐substrate interaction remain unclear. Here, we present oriented elliptical inverse opal structures for regulating the morphology and alignment of bone marrow‐derived MSCs. The inverse opal structures were made through a convenient bottom‐up approach of self‐assembly, which is facile and cost effective. MSCs cultured on the oriented structures were highly aligned and extended highly oriented thick lamellipodia. Moreover, the oriented substrates cracked along the lateral boundary of the cells, suggesting that a strong cell‐substrate interaction was induced by the response of MSCs to the oriented topography. These features of the oriented elliptical topography indicated their promising value in stem cell research and tissue engineering.     

Strategies for ensuring that regenerative cardiomyocytes function properly and in cooperation with the host myocardium

In developed countries, in which people have nutrient-rich diets, convenient environments, and access to numerous medications, the disease paradigm has changed. Nowadays, heart failure is one of the major causes of death. In spite of this, the therapeutic efficacies of medications are generally unsatisfactory. Although whole heart transplantation is ideal for younger patients with heart failure, many patients are deemed to be unsuitable for this type of surgery due to complications and/or age. The need for therapeutic alternatives to heart transplantation is great. Regenerative therapy is a strong option. For this purpose, several cell sources have been investigated, including intrinsic adult stem or progenitor cells and extrinsic pluripotent stem cells. Most intrinsic stem cells seem to contribute to a regenerative environment via paracrine factors and/or angiogenesis, whereas extrinsic pluripotent stem cells are unlimited sources of cardiomyocytes. In this review, we summarize the various strategies for using regenerative cardiomyocytes including our recent progressions: non-genetic approaches for the purification of cardiomyocytes and efficient transplantation. We expect that use of intrinsic and extrinsic stem cells in combination will enhance therapeutic effectiveness.     

The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.     

Chemical control of stem cell fate and developmental potential

Potential applications of stem cells in medicine range from their inclusion in disease modeling and drug discovery to cell transplantation and regenerative therapies. However, before this promise can be realized several obstacles must be overcome, including the control of stem cell differentiation, allogeneic rejection and limited cell availability. This will require an improved understanding of the mechanisms that govern stem cell potential and the development of robust methods to efficiently control their fate. Recently, a number of small molecules have been identified that can be used both in vitro and in vivo as tools to expand stem cells, direct their differentiation, or reprogram somatic cells to a more naive state. These molecules have provided a wealth of insights into the signaling and epigenetic mechanisms that regulate stem cell biology, and are already beginning to contribute to the development of effective treatments for tissue repair and regeneration.     

Gravitational field-flow fractionation of human hemopoietic stem cells

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.     

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n?=?1,001) were identified from cells cultured either with (n?=?857) or without (n?=?667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages

Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair.     

Recent Advances in BTK Inhibitors for the Treatment of Inflammatory and Autoimmune Diseases

Bruton’s tyrosine kinase (BTK) plays a crucial role in B-cell receptor and Fc receptor signaling pathways. BTK is also involved in the regulation of Toll-like receptors and chemokine receptors. Given the central role of BTK in immunity, BTK inhibition represents a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases. Great efforts have been made in developing BTK inhibitors for potential clinical applications in inflammatory and autoimmune diseases. This review covers the recent development of BTK inhibitors at preclinical and clinical stages in treating these diseases. Individual examples of three types of inhibitors, namely covalent irreversible inhibitors, covalent reversible inhibitors, and non-covalent reversible inhibitors, are discussed with a focus on their structure, bioactivity and selectivity. Contrary to expectations, reversible BTK inhibitors have not yielded a significant breakthrough so far. The development of covalent, irreversible BTK inhibitors has progressed more rapidly. Many candidates entered different stages of clinical trials; tolebrutinib and evobrutinib are undergoing phase 3 clinical evaluation. Rilzabrutinib, a covalent reversible BTK inhibitor, is now in phase 3 clinical trials and also offers a promising future. An analysis of the protein–inhibitor interactions based on published co-crystal structures provides useful clues for the rational design of safe and effective small-molecule BTK inhibitors.     

The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 µM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.     

Intervention with costimulatory pathways as a therapeutic approach for graft-versus-host disease

Graft-versus-host disease (GVHD) is mediated by mature donor T cells contained in the hematopoietic stem cell graft. During the development of GVHD, signaling through a variety of costimulatory receptors plays an important role in allogeneic T cell responses. Even though delivery of costimulatory signals is a prerequisite for full activation of donor T cells in the phase of their interactions with host APCs, their involvement with GVHD might occur over multiple stages. Like many other aspects of GVHD, promise of therapeutic interventions with costimulatory pathways has been gleaned from preclinical models. In this review, I summarize some of the advances in roles of costimulatory molecules in GVHD pathophysiology and discuss preclinical approaches that warrant further exploration in the clinic, focusing on novel strategies to delete pathogenic T cells.     

Recruitment of endogenous stem cells for tissue repair

The traditional concept of stem cell therapy envisions the isolation of stem cells from patients, propagation and differentiation in vitro, and subsequent re-injection of autologous cells into the patient. There are many problems associated with this paradigm, particularly during the in vitro manipulation process and the delivery and local retention of re-injected cells. An alternative paradigm that could be easier, safer, and more efficient, would involve attracting endogenous stem cells and precursor cells to the defect site for new tissue regeneration. Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin, exerts a strong chemoattractive effect on mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and induces migration of MSCs in vitro. However, HGF undergoes rapid proteolysis in vivo, which results in a very short lifetime of the bioactive cytokine. To maintain the therapeutic level of HGF at the defect site necessary for endogenous stem cell recruitment, sustained, long-term, and localized delivery of HGF is required. Thiol-modified glycosaminoglycans hyaluronan (HA) and heparin (HP), combined with modified gelatin (Gtn), have been crosslinked with poly(ethylene glycol) diacrylate (PEGDA) to afford semisynthetic ECM-like (sECM) hydrogels that can both provide controlled growth factor release and permit cell infiltration and proliferation. Herein we compare the use of different sECM compositions for controlled release of HGF and concomitant recruitment of human bone marrow MSCs into the scaffold in vitro. [Figure: see text].     

Design of Polymeric Culture Substrates to Promote Proangiogenic Potential of Stem Cells

Stem cells are a promising cell source for regenerative medicine due to their differentiation and self‐renewal capacities. In the field of regenerative medicine and tissue engineering, a variety of biomedical technologies have been tested to improve proangiogenic activities of stem cells. However, their therapeutic effect is found to be limited in the clinic because of cell loss, senescence, and insufficient therapeutic activities. To address this type of issue, advanced techniques for biomaterial synthesis and fabrication have been approached to mimic proangiogenic microenvironment and to direct proangiogenic activities. This review highlights the types of polymers and design strategies that have been studied to promote proangiogenic activities of stem cells. In particular, scaffolds, hydrogels, and surface topographies, as well as insight into their underlying mechanisms to improve proangiogenic activities are the focuses. The strategy to promote angiogenic activities of hMSCs by controlling substrate repellency is introduced, and the future direction is proposed.