Expression of CD45 in non-hematopoietic cells: implications in regenerative medicine and disease management.

CD45 plays a crucial role in the regulation of hematopoiesis. However, a comprehensive understanding of its role in non-hematopoietic cells is lacking. Several tissue precursors express CD45, indicating its crucial role in tissue regeneration. These precursors would fall prey to the recent therapies involving CD45 as a target. CD45+ double-positive tumor cells contribute to cancer progression, but whether CD45 is involved in the process needs to be investigated. Recently, we showed that aging induces CD45 expression in mesenchymal stromal cells and affects their differentiation potential. In this review, we, for the first time, unravel the important implications of the expression of CD45 in non-hematopoietic cells and provide novel insights into its potential therapeutic target in regenerative medicine and disease management.

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The symbiotic effect of osteoinductive extracellular vesicles and mineralized microenvironment on osteogenesis.

The increasing prevalence of bone-related diseases has raised concern about the need for an osteoinductive and mechanically stronger scaffold-based bone tissue engineering (BTE) alternative. A mineralized microenvironment, similar to the native bone microenvironment, is required in the scaffold to recruit and differentiate local mesenchymal stem cells at the bone defect site. Further, extracellular vesicles (EVs), pre-osteoblasts' secretome, contain osteoinductive cargo and have recently been exploited in bone regeneration. This work developed a cell-free and mechanically strong interpenetrating network-based scaffold for BTE by combining the action of osteoinductive EVs with a mineralized microenvironment. The MC3T3 (a pre-osteoblast cell line) is used as a source of EVs and as the target population. The optimal concentration of MC3T3-EVs was first determined to induce osteogenesis in target cells. The osteoinductive potential of the scaffold was estimated in vitro by osteogenesis-related markers like the alkaline phosphatase (ALP) enzyme and calcium content. The MC3T3-EVs cargo was also studied for osteoinductive signals such as ALP, calcium, and mRNA. The findings of this work indicated that MC3T3-EVs at a 90 µg/mL dose had significantly higher ALP activity than 0 µg/mL (1.47-fold), 10 µg/mL (1.41-fold), and 30 µg/mL (1.39-fold) EV-concentration on day 14. Further combination of the optimum dose of EVs with a mineralized microenvironment significantly enhanced ALP activity (1.5-fold) and mineralization (3.36-fold) as compared to the control group on day 7. EV cargo analysis revealed the presence of calcium, the ALP enzyme, and the mRNAs necessary for osteogenesis and angiogenesis. ALP activity was significantly boosted in the EV-containing target cells as early as day 1, and mineralization began on day 7 because MC3T3-EVs carry ALP enzymes and calcium as cargo. When osteoinductive EVs were combined with an osteoconductive mineralized microenvironment, osteogenesis was significantly enhanced in target cells at early time points. The interaction between osteoinductive EVs and the mineralized milieu facilitates the process of osteogenesis in the target cells and suggests a potential cell-free strategy for in vivo bone repair.

Signal transduction pathways alter the molecular cargo of extracellular vesicles: implications in regenerative medicine.

Extracellular vesicles (EVs) possess regenerative properties and are also considered as future vaccines. All types of cells secrete EVs; however, the amount of EVs secreted by the cells varies under various physiological as well as pathological states. Several articles have reviewed the molecular composition and potential therapeutic applications of EVs. Likewise, the 'sorting signals' associated with specific macromolecules have also been identified, but how the signal transduction pathways prevailing in the parent cells alter the molecular profile of the EVs or the payload they carry has not been sufficiently reviewed. Here, we have specifically discussed the implications of these alterations in the macromolecular cargo of EVs for their therapeutic applications in regenerative medicine.

Beyond animal models: revolutionizing neurodegenerative disease modeling using 3D in vitro organoids, microfluidic chips, and bioprinting.

Neurodegenerative diseases (NDs) are characterized by uncontrolled loss of neuronal cells leading to a progressive deterioration of brain functions. The transition rate of numerous neuroprotective drugs against Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, leading to FDA approval, is only 8-14% in the last two decades. Thus, in spite of encouraging preclinical results, these drugs have failed in human clinical trials, demonstrating that traditional cell cultures and animal models cannot accurately replicate human pathophysiology. Hence, in vitro three-dimensional (3D) models have been developed to bridge the gap between human and animal studies. Such technological advancements in 3D culture systems, such as human-induced pluripotent stem cell (iPSC)-derived cells/organoids, organ-on-a-chip technique, and 3D bioprinting, have aided our understanding of the pathophysiology and underlying mechanisms of human NDs. Despite these recent advances, we still lack a 3D model that recapitulates all the key aspects of NDs, thus making it difficult to study the ND's etiology in-depth. Hence in this review, we propose developing a combinatorial approach that allows the integration of patient-derived iPSCs/organoids with 3D bioprinting and organ-on-a-chip technique as it would encompass the neuronal cells along with their niche. Such a 3D combinatorial approach would characterize pathological processes thoroughly, making them better suited for high-throughput drug screening and developing effective novel therapeutics targeting NDs.

A comprehensive analysis of cell-autonomous and non-cell-autonomous regulation of myeloid leukemic cells: The prospect of developing novel niche-targeting therapies.

Leukemic cells (LCs) arise from the hematopoietic stem/and progenitor cells (HSCs/HSPCs) and utilize cues from the bone marrow microenvironment (BMM) for their regulation in the same way as their normal HSC counterparts. Mesenchymal stromal cells (MSCs), a vital component of the BMM promote leukemogenesis by creating a protective and immune-tolerant microenvironment that can support the survival of LCs, helping them escape chemotherapy, thereby resulting in the relapse of leukemia. Conversely, MSCs also induce apoptosis in the LCs and inhibit their proliferation by interfering with their self-renewal potential. This review discusses the work done so far on cell-autonomous (intrinsic) and MSCs-mediated non-cell-autonomous (extrinsic) regulation of myeloid leukemia with a special focus on the need to investigate the extrinsic regulation of myeloid leukemia to understand the contrasting role of MSCs in leukemogenesis. These mechanisms could be exploited to formulate novel therapeutic strategies that specifically target the leukemic microenvironment.

Cell-Instructive Mineralized Microenvironment Regulates Osteogenesis: A Growing SYMBIOSIS of Cell Biology and Biomaterials Engineering in Bone Tissue Regeneration.

One of the objectives of bone tissue engineering is to produce scaffolds that are biocompatible, osteoinductive, and mechanically equivalent to the natural extracellular matrix of bone in terms of structure and function. Reconstructing the osteoconductive bone microenvironment into a scaffold can attract native mesenchymal stem cells and differentiate them into osteoblasts at the defect site. The symbiotic relationship between cell biology and biomaterial engineering could result in composite polymers containing the necessary signals to recreate tissue- and organ-specific differentiation. In the current work, drawing inspiration from the natural stem cell niche to govern stem cell fate, the cell-instructive hydrogel platforms were constructed by engineering the mineralized microenvironment. This work employed two different hydroxyapatite delivery strategies to create a mineralized microenvironment in an alginate-PEGDA interpenetrating network (IPN) hydrogel. The first approach involved coating of nano-hydroxyapatite (nHAp) on poly(lactide-co-glycolide) microspheres and then encapsulating the coated microspheres in an IPN hydrogel for a sustained release of nHAp, whereas the second approach involved directly loading nHAp into the IPN hydrogel. This study demonstrate that both direct encapsulation and a sustained release approach showed enhanced osteogenesis in target-encapsulated cells; however, direct loading of nHAp into the IPN hydrogel increased the mechanical strength and swelling ratio of the scaffold by 4.6-fold and 1.14-fold, respectively. In addition, the biochemical and molecular studies revealed improved osteoinductive and osteoconductive potential of encapsulated target cells. Being less expensive and simple to perform, this approach could be beneficial in clinical settings.

Dexamethasone release pattern via a three-dimensional system for effective bone regeneration.

For over a decade, dexamethasone (DEX) has been used for bone regenerative and anti-inflammatory purposes. It has also shown promise for inducing bone regeneration by using it as component of osteoinductive differentiation medium, particularly forin vitroculture models. Despite its osteoinductive properties, its use is limited due to its associated cytotoxicity, particularly when used at higher concentrations. DEX has adverse effects when taken orally; thus, it is best to use it in a targeted manner. Even when given locally, the pharmaceutical should be distributed in a controlled manner based on the needs of the wounded tissue. However, because drug activity is assessed in two-dimensional (2D) circumstances and the target tissue is a three-dimensional (3D) structure, assessing DEX activity and dosage in a 3D milieu is critical for bone tissue development. The current review examines the advantages of a 3D approach over traditional 2D culture methods and delivery devices for controlled DEX delivery, particularly for bone repair. Further, this review explores the latest advancement and challenges in biomaterial-based therapeutic delivery approaches for bone regeneration. This review also discusses possible future biomaterial-based strategies to study efficient DEX delivery.

Priming mesenchymal stromal cells with neurotrophic factors boosts the neuro-regenerative potential of their secretome.

Aim: To explore the neuroprotective potential of the secretome (conditioned medium, CM) derived from neurotrophic factors-primed mesenchymal stromal cells (MSCs; primed CM) using an endoplasmic reticulum (ER) stress-induced in vitro model system. Methods: Establishment of ER-stressed in vitro model, immunofluorescence microscopy, real-time PCR, western blot. Results: Exposure of ER-stressed Neuro-2a cells to the primed-CM significantly restored the neurite outgrowth parameters and improved the expression of neuronal markers like Tubb3 and Map2a in them compared with the naive CM. Primed CM also suppressed the induction of apoptotic markers Bax and Sirt1, inflammatory markers Cox2 and NF-κB, and stress kinases such as p38 and SAPK/JNK in the stress-induced cells. Conclusion: The secretome from primed MSCs significantly restored ER stress-induced loss of neuro-regenesis.

Endoplasmic reticulum (ER) stress-mediated accumulation of misfolded protein is one of the causes involved in the onset of several neurodegenerative diseases (ND). Under physiological conditions, ER stress activates the unfolded protein response (UPR) that repairs the misfolded proteins. However, if the ER stress becomes chronic, the UPR fails to repair the misfolded proteins leading to disease conditions such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, etc. Most in vitro systems are based on the infliction of acute ER stress on the target cells, which kills them, and thus, are not physiologically relevant models, as their neuro-regeneration is not possible. Here, we have developed a physiologically relevant in vitro model system, wherein we exposed Neuro-2a cells to an ER stress inducer which significantly affected their neuro-regenesis without killing them. These dysfunctional cells were then used to assess the effect of secretome (conditioned medium, CM) derived from mesenchymal stromal cells (MSCs) primed or not with neurotrophic factors. We found that priming of MSCs with neurotrophic factors enhances their neuroprotective potential. We demonstrate that when primed CM is given either as a single dose or in multiple doses, it restores neuro-regenesis and protects the stressed Neuro-2a cells from cell death. More importantly, the restoration of neuro-regenesis by primed CM is significantly higher as compared with the naive CM. These results clearly underscore the importance of priming the MSCs with neurotrophic factors for developing more effective therapeutic strategies to combat ND.

Secretome of Young Mesenchymal Stromal Cells Rejuvenates Aged Mesenchymal Stromal Cells by Normalizing Their Phenotype and Restoring Their Differentiation Profile.

During aging, the proliferation and differentiation ability of mesenchymal stem/stromal cells (MSCs) gets affected, and hence, aged MSCs are not preferred for regenerative purposes. Rapid identification of aging-associated changes within MSCs and the mechanistic pathways involved are necessary to determine optimal cell sources to treat musculoskeletal disorders in older patients. In the present study, we have identified a set of phenotypic markers, namely downregulated expression of CD90 and upregulated expression of CD45, as age-defining markers for the bone marrow-derived MSCs. We also show that these phenotypic changes in aged MSCs correlate with their aging-mediated differentiation defects. We find that oxidative stress signaling leading to the activation of nuclear factor kappa B (NF-κB) plays an essential role in altering the phenotype and differentiation ability of the aged MSCs. We further show that treatment of aged MSCs with the conditioned medium (CM) derived from young MSCs (young-CM) restored their phenotype and differentiation potential to the young-like by ameliorating activation of NF-κB signaling in them. Similar changes could also be achieved by using an inhibitor of NF-κB signaling, showing that oxidative stress-induced NF-κB activation is the causative factor in the aging of MSCs. Additionally, we show that treating young MSCs with hydrogen peroxide mimics all the aging-mediated changes in them, underscoring the involvement of oxidative stress in the aging of MSCs. Overall, our data suggest that the altered expression of CD90 and CD45 surface markers can be used as a primary screen to identify the onset of aging in the MSCs, which can be quickly reversed by their in vitro treatment with young-CM or NF-κB inhibitor. Our study also puts the phenotypic characterization of MSCs in a clinical perspective.

Cell-intrinsic factors governing quiescence vis-à-vis activation of adult hematopoietic stem cells.

Hematopoiesis is a highly complex process, regulated by both intrinsic and extrinsic factors. Often, these two regulatory arms work in tandem to maintain the steady-state condition of hematopoiesis. However, at times, certain intrinsic attributes of hematopoietic stem cells (HSCs) override the external stimuli and dominate the outcome. These could be genetic events like mutations or environmentally induced epigenetic or transcriptomic changes. Since leukemic stem cells (LSCs) share molecular pathways that also regulate normal HSCs, identifying specific, dominantly acting intrinsic factors could help in the development of novel therapeutic approaches. Here we have reviewed such dominantly acting intrinsic factors governing quiescence vis-à-vis activation of the HSCs in the face of external forces acting on them. For brevity, we have restricted our review to the articles dealing with adult HSCs of human and mouse origin that have been published in the last 10 years. Hematopoietic stem cells (HSCs) are closely associated with various stromal cells in their microenvironment and, thus, constantly receive signaling cues from them. The illustration depicts some dominantly acting intrinsic or cell-autonomous factors operative in the HSCs. These fall into various categories, such as epigenetic regulators, transcription factors, cell cycle regulators, tumor suppressor genes, signaling pathways, and metabolic regulators, which counteract the outcome of extrinsic signaling exerted by the HSC niche.

A chimeric feeder comprising transforming growth factor beta 1- and basic fibroblast growth factor-primed bone marrow-derived mesenchymal stromal cells suppresses the expansion of hematopoietic stem and progenitor cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) physically associate with the hematopoietic stem cells (HSCs), forming a unique HSC niche. Owing to this proximity, the signaling mechanisms prevailing in the BMSCs affect the fate of the HSCs. In addition to cell-cell and cell-extracellular matrix interactions, various cytokines and growth factors present in the BM milieu evoke signaling mechanisms in the BMSCs. Previously, I have shown that priming of human BMSCs with transforming growth factor ß1 (TGFß1), a cytokine consistently found at active sites of hematopoiesis, boosts their hematopoiesis-supportive ability. Basic fibroblast growth factor (bFGF), another cytokine present in the marrow microenvironment, positively regulates hematopoiesis. Hence, I examined whether priming human BMSCs with bFGF improves their hematopoiesis-supportive ability. I found that bFGF-primed BMSCs stimulate hematopoiesis, as seen by a significant increase in colony formation from the bone marrow cells briefly interacted with them and the extensive proliferation of CD34+ HSCs cocultured with them. However, contrary to my expectation, I found that chimeric feeders comprising a mixture of TGF-primed and bFGF-primed BMSCs exerted a suppressive effect. These data demonstrate that though the TGF- and bFGF-primed BMSCs exert a salutary effect on hematopoiesis when used independently, they exert a suppressive effect when presented as a chimera. These findings suggest that the combinatorial effect of various priming agents and cytokines on the functionality of BMSCs toward the target tissues needs to be critically evaluated before they are clinically applied.

Well-orchestrated physico-chemical and biological factors for enhanced secretion of osteogenic and angiogenic extracellular vesicles by mesenchymal stem cells in a 3D culture format.

The secretome of mesenchymal stem cells (MSCs) is being studied for its regenerative potential for the treatment of various disorders, including bone diseases. However, mimicking the physiological parameters of native bone could further improve MSCs' secretory profile. The proteomic analysis revealed that MSCs have a diverse secretory profile depending on the cell formats used to grow them, such as two-dimensional (2D) or three-dimensional (3D) microenvironments. Stem cells are given biochemical and biophysical stimuli in a 3D milieu that mimics in vivo situations. Compared to the gold standard monolayer culture, extracellular vesicles (EVs) released under 3D conditions improved the EV cargo numerically and qualitatively. The higher requirements of EVs in clinical trials with consistent therapeutic potential are challenging. This review discusses the impact of cell culture formats on the regenerative potential of MSCs, specifically in bone regeneration. The poor yield and heterogeneity issues have hampered the therapeutic usage of EVs. Therefore, this review further explores various engineering approaches that could enhance EVs' scalability from MSCs and their therapeutic effectiveness beyond their native utility in bone tissue regeneration. This review also highlights some of the upcoming 3D approaches/models that might be useful for the enhanced secretion of therapeutic EVs from stem cells. Finally, we discuss possible future directions and conclusions in this domain.

Clinical applications of pluripotent stem cells and their derivatives: current status and future perspectives.

Pluripotent stem cells (PSCs) can differentiate into specific cell types and thus hold great promise in regenerative medicine to treat certain diseases. Hence, several studies have been performed harnessing their salutary properties in regenerative medicine. Despite several challenges associated with the clinical applications of PSCs, worldwide efforts are harnessing their potential in the regeneration of damaged tissues. Several clinical trials have been performed using PSCs or their derivatives. However, the delay in publishing the data obtained in the trials has led to a lack of awareness about their outcomes, resulting in apprehension about cellular therapies. Here, the authors review the published papers containing data from recent clinical trials done with PSCs. PSC-derived extracellular vesicles hold great potential in regenerative therapy. Since published papers containing the data obtained in clinical trials on PSC-derived extracellular vesicles are not available yet, the authors have reviewed some of the pre-clinical work done with them.

Embryonic stem cells (ESCs) can make all types of cells in the body. Likewise, induced pluripotent stem cells (iPSCs), which are laboratory-generated counterparts of ESCs, possess similar properties. ESCs and iPSCs have immense application in regenerative medicine, as they can be the only cure for certain diseases and conditions that are incurable with currently available treatments; however, several challenges remain. Notably, many clinical trials using these cells or their products are going on globally. However, due to the extensive time frame required to complete the clinical trials and publish the data obtained, the outcomes of these trials do not reach the general population. This delay in information flow to the public domain creates apprehension about cellular therapy. Here, the authors have reviewed recent publications documenting the results obtained in the clinical trials done with ESCs and iPSCs (together referred to as pluripotent stem cells). The vesicles (called extracellular vesicles) secreted by pluripotent stem cells also have great regenerative potential. Since published papers containing the results obtained in clinical trials done with these vesicles are not available yet, the authors have reviewed some pre-clinical work done on them.

Mesenchymal stromal cells-derived secretome protects Neuro-2a cells from oxidative stress-induced loss of neurogenesis.

Neurodegenerative diseases (ND) are characterized by debilitating medical conditions that principally affect the neuronal cells in the human brain. One of the major reasons that there are no effective drugs for the treatment of ND is because researchers face technical challenges while conducting studies to understand the molecular mechanism behind ND. Although various studies have established in vitro neurodegenerative model systems, we feel that these model systems are not physiologically relevant, as they do not mimic the in vivo situation of chronic insult. Therefore, the primary aim of this study was to establish an in vitro neurodegenerative model system by inducing oxidative stress in such a way that the neuronal cells remain viable, but lose their structural and functional characteristics. Using a murine neuroblastoma cell line, Neuro-2a, we demonstrate that induction of oxidative stress significantly affects various neurite outgrowth parameters and reduces the expression of neuronal and autophagy markers without causing apoptosis in them. Previously, we have discussed the possible therapeutic applications of mesenchymal stromal cells (MSCs) and their secretome in the treatment of ND. Here, using two distinct approaches, we show that when Neuro-2a cells subjected to oxidative stress are exposed to MSC-derived conditioned medium (secretome), they exhibit a significant improvement in various neuronal parameters and in the expression of neuronal markers. Overall, our findings support the salutary role of MSC-derived secretome in rescuing the oxidative stress-induced loss of neurogenesis using a physiologically relevant in vitro model system. Our data underscore the propensity of the MSC-secretome in reversing ND.

The Intercellular Communication Between Mesenchymal Stromal Cells and Hematopoietic Stem Cells Critically Depends on NF-κB Signalling in the Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) regulate the fate of the hematopoietic stem cells (HSCs) through both cell-cell interactions and paracrine mechanisms involving multiple signalling pathways. We have previously shown that co-culturing of HSCs with CoCl2-treated MSCs expands functional HSCs. While performing these experiments, we had observed that the growth of CoCl2-treated MSCs was significantly stunted. Here, we show that CoCl2-treated MSCs possess activated NF-κB signalling pathway, and its pharmacological inhibition significantly relieves their growth arrest. Most interestingly, we found that pharmacological inhibition of NF-κB pathway in both control and CoCl2-treated MSCs completely blocks their intercellular communication with the co-cultured hematopoietic stem and progenitor cells (HSPCs), resulting in an extremely poor output of hematopoietic cells. Mechanistically, we show that this is due to the down-regulation of adhesion molecules and various HSC-supportive factors in the MSCs. This loss of physical interaction with HSPCs could be partially restored by treating the MSCs with calcium ionophore or calmodulin, suggesting that NF-κB regulates intracellular calcium flux in the MSCs. Importantly, the HSPCs co-cultured with NF-κB-inhibited-MSCs were in a quiescent state, which could be rescued by re-culturing them with untreated MSCs. Our data underscore a critical requirement of NF-κB signalling in the MSCs in intercellular communication between HSCs and MSCs for effective hematopoiesis to occur ex vivo. Our data raises a cautionary note against excessive use of anti-inflammatory drugs targeting NF-κB.

Granulocytes Negatively Regulate Secretion of Transforming Growth Factor ß1 by Bone Marrow Mononuclear Cells via Secretion of Erythropoietin Receptors in the Milieu.

In my previous study, I demonstrated that bone marrow-derived mononuclear cells (BM MNCs) secrete copious amounts of Transforming Growth Factor ß1 (TGFß1) in response to erythropoietin (EPO). In this study, I investigated the principal cell type involved in the process. I found that a large percentage of various marrow cells, but not their mature counterparts present in the peripheral blood, express EPO-receptors (EPO-R). Cell depletion experiments showed that depletion of Glycophorin positive erythroblasts and CD41+ megakaryocytes - the prime suspects - did not affect the EPO-mediated TGFß1 secretion by the BM MNCs. However, individual depletion of CD2+ T lymphocytes, CD14+ monocyte/macrophages, and CD19+ B cells affected the TGFß1 secretion by EPO-primed MNCs: depletion of CD2+ cells had the most striking effect. Unexpectedly, and most interestingly, depletion of CD15+ granulocytes led to a significant increase in the TGFß1 secretion by both naïve and EPO-primed BM MNCs, suggesting that these cells negatively regulate the process. Mechanistically, I show that the CD15+ cells exert this regulatory effect via secretion of both full-length and soluble EPO-R in the milieu. Overall my results, for the first time, unravel an in-built regulatory mechanism prevailing in the BM microenvironment that regulates the secretion of TGFß1 by controlling EPO-EPO-R interaction.My data could be relevant in understanding the pathophysiology of several conditions associated with deregulated production of TGFß1 in the marrow compartment.

Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability.

The therapeutic value of mesenchymal stromal cells (MSCs) for various regenerative medicine applications, including hematopoietic stem cell transplantations (HSCT), has been well-established. Owing to their small numbers in vivo, it becomes necessary to expand them in vitro, which leads to a gradual loss of their regenerative capacity. Stress-induced mitogen-activated protein kinase p38 (p38 MAPK) signaling has been shown to compromise the MSC functions. Therefore, we investigated whether pharmacological inhibition of p38 MAPK signaling rejuvenates the cultured MSCs and boosts their functionality. Indeed, we found that the ex vivo expanded MSCs show activated p38 MAPK signaling and exhibit increased oxidative stress. These MSCs show a decreased ability to secrete salutary niche factors, thereby compromising their ability to support hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation. We, therefore, attempted to rejuvenate the cultured MSCs by pharmacological inhibition of p38 MAPK - a strategy broadly known as "priming of MSCs". We demonstrate that priming of MSCs with a p-38 MAPK inhibitor, PD169316, boosts their niche-supportive functions via upregulation of various HSC-supportive transcription factors. These primed MSCs expand multipotent HSCs having superior homing and long-term reconstitution ability. These findings shed light on the significance of non-cell-autonomous mechanisms operative in the hematopoietic niche and point towards the possible use of pharmacological compounds for rejuvenation of ex vivo cultured MSCs. Such approaches could improve the outcome of regenerative therapies involving in vitro cultured MSCs.

Involvement of extracellular vesicles in aging process and their beneficial effects in alleviating aging-associated symptoms.

Aging is a gradual and unavoidable physiological phenomenon that manifests in the natural maturation process and continues to progress from infanthood to adulthood. Many elderly people suffer from aging-associated hematological and nonhematological disorders. Recent advances in regenerative medicine have shown new revolutionary paths of treating such diseases using stem cells; however, aging also affects the quality and competence of stem and progenitor cells themselves and ultimately directs their death or apoptosis and senescence, leading to a decline in their regenerative potential. Recent research works show that extracellular vesicles (EVs) isolated from different types of stem cells may provide a safe treatment for aging-associated disorders. The cargo of EVs comprises packets of information in the form of various macromolecules that can modify the fate of the target cells. To harness the true potential of EVs in regenerative medicine, it is necessary to understand how this cargo contributes to the rejuvenation of aged stem and progenitor populations and to identify the aging-associated changes in the macromolecular profile of the EVs themselves. In this review, we endeavor to summarize the current knowledge of the involvement of EVs in the aging process and delineate the role of EVs in the reversal of aging-associated phenotypes. We have also analyzed the involvement of the molecular cargo of EVs in the generation of aging-associated disorders. This knowledge could not only help us in understanding the mechanism of the aging process but could also facilitate the development of new cell-free biologics to treat aging-related disorders in the future.

Differentiated Cells Derived from Hematopoietic Stem Cells and Their Applications in Translational Medicine.

Hematopoietic stem cells (HSCs) and their development are one of the most widely studied model systems in mammals. In adults, HSCs are predominantly found in the bone marrow, from where they maintain homeostasis. Besides bone marrow and mobilized peripheral blood, cord blood is also being used as an alternate allogenic source of transplantable HSCs. HSCs from both autologous and allogenic sources are being applied for the treatment of various conditions like blood cancers, anemia, etc. HSCs can further differentiate to mature blood cells. Differentiation process of HSCs is being extensively studied so as to obtain a large number of pure populations of various differentiated cells in vitro so that they can be taken up for clinical trials. The ability to generate sufficient quantity of clinical-grade specialized blood cells in vitro would take the field of hematology a step ahead in translational medicine.

Hydrogel-Assisted 3D Model to Investigate the Osteoinductive Potential of MC3T3-Derived Extracellular Vesicles.

Effective and rapid regeneration of bone defects often pose substantial challenges in severe accidental injuries and disabilities occurring due to diseases and/or advanced age, especially in patients having reduced tissue regeneration competence. The success of mesenchymal stromal cell (MSC)-based research strategies in improving bone regeneration was hampered not only due to the limited knowledge of therapeutic actions of MSCs but also due to difficulties as well as expenses related to cell manufacturing and time taken for ethical approvals for clinical use of living cells and engineered tissues. The recent trend indicated that there is a shift from the direct usage of MSCs toward the application of paracrine factors and extracellular vesicles (EVs) isolated from their MSC secretome in bone tissue regeneration. This shift has directed research into the development of "cell-free" therapeutics, which could be a better alternative due to its several advantages over the use of their parental MSCs. Furthermore, accumulating evidence suggested that the 3D microenvironment influences the paracrine effects of MSCs. Although the osteogenic role of EVs has been explored recently, the current study showed, for the first time, that encapsulation of EVs along with MC3T3 cells in a 3D hydrogel-assisted culture with a distinct porous microenvironment having meso and macro (0.05-200 µm) pore size distribution resulted in an improved osteogenic response in vitro. The present work was primarily focused on investigating the influence of EVs isolated under distinct priming conditions to enhance the osteogenic potential. In addition, in the current work, the osteogenic ability of different types of EVs (microvesicles and exosomes) and total EVs isolated at different time points was also examined when encapsulated with MC3T3 cells in an alginate gel. Using various biochemical assays, such as alkaline phosphatase (ALP) production and calcium secretion, it was observed that both microvesicles and exosomes collected from MC3T3 cells independently had osteogenic potential; however, their collective activity was found to be superior. We further showed that EVs induce an early osteogenic response in MC3T3 cells as indicated by ALP and calcium secretion at a much earlier time point, compared to the controls. Our data suggested that this 3D hydrogel-assisted system provides close proximity of cells and EVs, and thus, mimics the in vivo scenario, making it clinically useful for bone tissue engineering.