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.

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 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.

Toxic implications of silver nanoparticles on the central nervous system: A systematic literature review.

Silver nanoparticles have many medical and commercial applications, but their effects on human health are poorly understood. They are used extensively in products of daily use, but little is known about their potential neurotoxic effects. A xenobiotic metal, silver, has no known physiological significance in the human body as a trace metal. Biokinetics of silver nanoparticles indicates its elimination from the body via urine and feces route. However, a substantial amount of evidence from both in vitro and in vivo experimental research unequivocally establish the fact of easier penetration of smaller nanoparticles across the blood-brain barrier to enter in brain and thereby interaction with cellular components to induce neurotoxic effects. Toxicological effects of silver nanoparticles rely on the degree of exposure, particle size, surface coating, and agglomeration state as well as the type of cell or organism used to evaluate its toxicity. This review covers pertinent facts and the present state of knowledge about the neurotoxicity of silver nanoparticles reviewing the impacts on oxidative stress, neuroinflammation, mitochondrial function, neurodegeneration, apoptosis, and necrosis. The effect of silver nanoparticles on the central nervous system is a topic of growing interest and concern that requires immediate consideration.

Cryopreservation of mesenchymal stromal cell-derived extracellular vesicles using trehalose maintains their ability to expand hematopoietic stem cells in vitro.

The multitude of clinical trials using mesenchymal stromal cells (MSCs) has underscored their significance as a promising cell source for regenerative therapies. Most studies have however shown that MSCs get entrapped into the microvasculature of lungs, liver and spleen. In addition to intercellular communication, MSCs exert their effects in a paracrine manner by secretion of extracellular vesicles (EVs). The therapeutic effects of MSC-derived EVs have been examined in several diseases such as hepatic failure, liver injury, hematopoiesis etc. Therefore, optimization of cryopreservation strategies for the long-term storage of functional EVs could help in the development of off-the-shelf biologics. The aim of this study was to develop an optimal cryopreservation strategy for the efficient storage of both types of EVs - Microvesicles (MVs) and exosomes, independently, and to further examine the effect of the cryopreserved EVs on the ex vivo expansion of HSCs. MVs and exosomes were separately cryopreserved at different temperatures using PBS or PBS supplemented with trehalose (pTRE), and these cryopreserved EVs were then assessed for their functionality after revival. We found that addition of trehalose during cryopreservation helped in maintaining the morphology and functionality of the EVs, as assessed by their HSC-supportive potential, ability to expand phenotypically defined HSCs and ability to maintain the chemotactic migration potential of the HSCs co-cultured with them. This strategy could prove to be beneficial for facilitating the use of EVs as cell-free ready-to-use biologics for the ex vivo expansion of HSCs and in regenerative medicine.

Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells.

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

Mesenchymal stromal cell-derived extracellular vesicles as cell-free biologics for the ex vivo expansion of hematopoietic stem cells.

Hematopoietic stem cell transplantation (HSCT) is the ultimate choice of treatment for patients with hematological diseases and cancer. The success of HSCT is critically dependent on the number and engraftment efficiency of the transplanted donor hematopoietic stem cells (HSCs). Various studies show that bone marrow-derived mesenchymal stromal cells (MSCs) support hematopoiesis and also promote ex vivo expansion of HSCs. MSCs exert their therapeutic effect through paracrine activity, partially mediated through extracellular vesicles (EVs). Although the physiological function of EVs is not fully understood, inspiring findings indicate that MSC-derived EVs can reiterate the hematopoiesis, supporting the ability of MSCs by transferring their cargo containing proteins, lipids, and nucleic acids to the HSCs. The activation state of the MSCs or the signaling mechanism that prevails in them also defines the composition of their EVs, thereby influencing the fate of HSCs. Modulating or preconditioning MSCs to achieve a specific composition of the EV cargo for the ex vivo expansion of HSCs is, therefore, a promising strategy that can overcome several challenges associated with the use of naïve/unprimed MSCs. This review aims to speculate upon the potential role of preconditioned/primed MSC-derived EVs as "cell-free biologics," as a novel strategy for expanding HSCs in vitro.

Membrane trafficking RNA interference screen identifies a crucial role of the clathrin endocytic pathway and ARP2/3 complex for Japanese encephalitis virus infection in HeLa cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is one of the leading global causes of virus-induced encephalitis. The infectious life-cycle of viruses is heavily dependent on the host membrane trafficking network. Here, we have performed a RNA-interference-based screen using a siRNA panel targeting 136 membrane trafficking proteins to identify the key regulators of JEV infection in HeLa cells. We identified 35 proteins whose siRNA depletion restricts JEV replication by over twofold. We observe that JEV infection in HeLa cells is largely dependent on components of the clathrin-mediated endocytic (CME) pathway. Proteins involved in actin-filament-based processes, specifically CDC42 and members of the ARP2/3 complex are crucial for establishment of infection. Pharmacological pertubations of actin polymerization, a small molecule inhibitor of actin nucleation by the ARP2/3 complex - CK-548 - and the inhibitor of neural Wiskott-Aldrich syndrome proteins- Wiskostatin- inhibited JEV replication, highlighting the important role of the dynamic actin network. Other proteins involved in cargo-recognition for CME and endomembrane system organization were also validated as essential host factors for virus replication.

AKT Signaling Prevailing in Mesenchymal Stromal Cells Modulates the Functionality of Hematopoietic Stem Cells via Intercellular Communication.

The AKT pathway plays an important role in various aspects of stem cell biology. However, the consequences of constitutive activation of AKT in mesenchymal stromal cells (MSCs) on the fate of hematopoietic stem cells (HSCs) were unknown. Here, we show that bone marrow-derived MSCs expressing a constitutively active AKT1 expand HSCs, but severely affect their functionality. Conversely, stromal cells with silenced AKT1 limit HSC proliferation, but boost their functionality. These effects were related to differential modulation of several important regulatory genes, in both, the cocultured HSCs and in the stromal cells themselves. The detrimental effect of stromal cells with constitutively activated AKT1 involved dynamin-dependent endocytosis, whereas the salutary effect of stromal cells devoid of AKT1 was mediated via GAP junctions. Constitutive activation of AKT1 led to deregulated formation of GAP junctions in the stromal cells, which consequently exhibited strikingly increased intercellular transfer of molecular cargo to the HSCs. Conversely, stromal cells with silenced AKT1 exhibited normal intercellular arrangement of GAP junctions at appositional membrane areas, and did not show aberrant intercellular transfer. Micro-vesicles isolated from conditioned media of the stromal cells not only mimicked the effect of these cells, but also showed stronger effects. This is perhaps the first report demonstrating that AKT1 signaling prevailing in the MSCs regulates HSC functionality through various intercellular communication mechanisms. These findings could have important implications in the use of MSCs in regenerative medicine. Stem Cells 2016;34:2354-2367.

Chondroitin Sulfate and Hyaluronic Acid-Based PolyHIPE Scaffolds for Improved Osteogenesis and Chondrogenesis In Vitro.

Osteochondral damage, affecting the articular cartilage and the underlying subchondral bone, presents significant challenges in clinical treatment. Such defects, commonly seen in knee and ankle joints, vary from small localized lesions to larger defects. Current medical therapies encounter several challenges, such as donor shortages, drug side effects, high costs, and rejection problems, often resulting in only temporary relief. Highly porous emulsion-templated polymers (polyHIPEs) offer numerous potential benefits in the fabrication of scaffolds for tissue engineering and regenerative medicine. Polymeric scaffolds synthesized using a high internal phase emulsion (HIPE) technique, called PolyHIPEs, involve polymerizing a continuous phase surrounding a dispersed internal phase to form a solid, foam-like structure. A dense, porous design encourages cell ingrowth, nutrient delivery, and waste disposal from the scaffold, mimicking the cells' natural microenvironment. This study used hydroxyethyl methacrylate (HEMA) and acrylamide (AAM) polyHIPE scaffolds combined with extracellular matrix (ECM) components of the tissue, such as methacrylated hyaluronic acid (MHA) and methacrylated chondroitin sulfate (MCS), to prepare polyHIPE scaffolds. The mouse preosteoblast MC3T3-E1 cells and primary rat chondrocytes (harvested from male Wistar rats) were seeded on the scaffolds and cultured for 21 days to assess the osteogenesis and chondrogenesis in vitro. When compared to the AAM-MHA and AAM-MCS groups at day 21, scaffold groups HEMA-MHA and HEMA-MCS showed a significant rise in alkaline phosphatase (ALP) and calcium content. Chondrogenic markers such as glycosaminoglycan (GAG) and hydroxyproline were also assessed over a 21-day time point. On day 21, it was found that GAG and hydroxyproline production were considerably higher in the HEMA-MHA and HEMA-MCS scaffolds than in the AAM-MHA and AAM-MCS scaffolds. The overall studies showed that polyHIPE monolith scaffolds could favor cell adherence, survival ability, proliferation, differentiation, and ECM formation over 21 days. Thus, incorporating ECM components enhanced osteogenesis and chondrogenesis in vitro and can be further used as tissue repair models.

Size, Surface Properties, and Ion Release of Zinc Oxide Nanoparticles: Effects on Cytotoxicity, Dopaminergic Gene Expression, and Acetylcholinesterase Inhibition in Neuronal PC-12 Cells.

The extensive applications of zinc oxide nanoparticles (ZnO NPs) have resulted in a substantial risk of human exposure. However, the knowledge of the toxicity of these NPs in the nervous system is still limited. A comparative analysis of ZnO NPs of various sizes and NPs of the same size, with and without surface coating, and the potential role of released zinc ions is yet to be thoroughly explored. As a result, we have studied the cellular toxicity of two different-sized ZnO NPs, ZnO-22 (22 nm) and ZnO-43 (43 nm), and NPs with similar size but with polyvinylpyrrolidone coating (ZnO-P, 45 nm). The findings from our study suggested a time-, size-, and surface coating-dependent cytotoxicity in PC-12 cells at a concentration ≥ 10 µg/ml. ZnO NP treatment significantly elevated reactive oxygen and reactive nitrogen species, thereby increasing oxidative stress. The exposure of ZnO-22 and ZnO-43 significantly upregulated the expression of monoamine oxidase-A and downregulated the α-synuclein gene expression associated with the dopaminergic system. The interaction of NPs enzymes in the nervous system is also hazardous. Therefore, the inhibition activity of acetylcholinesterase enzyme was also studied for its interaction with these NPs, and the results indicated a dose-dependent inhibition of enzyme activity. Particle size, coating, and cellular interactions modulate ZnO NP's cytotoxicity; smaller sizes enhance cellular uptake and reactivity, while coating reduces cytotoxicity by limiting direct cell contact and potentially mitigating oxidative stress. Furthermore, the study of released zinc ions from the NPs suggested no significant contribution to the observed cytotoxicity compared to the NPs.

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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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.

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.

Comprehensive Analysis of Titanium Oxide Nanoparticle Size and Surface Properties on Neuronal PC-12 Cells: Unraveling Cytotoxicity, Dopaminergic Gene Expression, and Acetylcholinesterase Inhibition.

Titanium oxide nanoparticles can penetrate the blood-brain barrier, infiltrate the central nervous system, and induce neurotoxicity. One of the most often utilized nanoparticles has been investigated for their neurotoxicity in many studies. Nonetheless, there remains an unexplored aspect regarding the comparative analysis of particles varying in size and nanoparticles of identical dimensions, both with and devoid of surface coating. In the current study, we synthesized two differently sized nanoparticles, TiO2-10 (10 nm) and TiO2-22 (22 nm), and nanoparticles of the same size but with a polyvinylpyrrolidone surface coating (TiO2-PVP, 22 nm) and studied their toxic effects on neural PC-12 cells. The results highlighted significant dose- and time-dependent cytotoxicity at concentrations ≥10 µg/mL. The exposure of TiO2 nanoparticles significantly elevated reactive oxygen and nitrogen species levels, IL-6 and TNF-α levels, altered the mitochondrial membrane potential, and enhanced apoptosis-related caspase-3 activity, irrespective of size and surface coating. The interaction of the nanoparticles with acetylcholinesterase enzyme activity was also investigated, and the results revealed a dose-dependent suppression of enzymatic activity. However, the gene expression studies indicated no effect on the expression of all six genes associated with the dopaminergic system upon exposure to 10 µg/mL for any nanoparticle. The results demonstrated no significant difference between the outcomes of TiO2-10 and TiO2-22 NPs. However, the polyvinylpyrrolidone surface coating was able to attenuate the neurotoxic effects. These findings suggest that as the TiO2 nanoparticles get smaller (towards 0 nm), they might promote apoptosis and inflammatory reactions in neural cells via oxidative stress, irrespective of their size.

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.

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.

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.

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.