Use of priming strategies to advance the clinical application of mesenchymal stromal/stem cell-based therapy

Mesenchymal stromal/stem cells(MSCs)have garnered significant attention in the field of regenerative medicine due to their remarkable therapeutic potential.MSCs play a pivotal role in maintaining tissue homeostasis and possess diverse functions in tissue repair and recovery in various organs.These cells are charac-terized by easy accessibility,few ethical concerns,and adaptability to in vitro cultures,making them a valuable resource for cell therapy in several clinical conditions.Over the years,it has been shown that the true therapeutic power of MSCs lies not in cell engraftment and replacement but in their ability to produce critical paracrine factors,including cytokines,growth factors,and exosomes(EXOs),which modulate the tissue microenvironment and facilitate repair and regeneration processes.Consequently,MSC-derived products,such as condi-tioned media and EXOs,are now being extensively evaluated for their potential medical applications,offering advantages over the long-term use of whole MSCs.However,the efficacy of MSC-based treatments varies in clinical trials due to both intrinsic differences resulting from the choice of diverse cell sources and non-standardized production methods.To address these concerns and to enhance MSC therapeutic potential,researchers have explored many priming strategies,including exposure to inflammatory molecules,hypoxic conditions,and three-dimensional culture techniques.These approaches have optimized MSC secretion of functional factors,empowering them with enhanced immunomodulatory,angiogenic,and regenerative properties tailored to specific medical conditions.In fact,various priming strategies show promise in the treatment of numerous diseases,from immune-related disorders to acute injuries and cancer.Currently,in order to exploit the full therapeutic potential of MSC therapy,the most important challenge is to optimize the modulation of MSCs to obtain adapted cell therapy for specific clinical disorders.In other words,to unlock the complete potential of MSCs in regenerative medicine,it is crucial to identify the most suitable tissue source and develop in vitro manipulation protocols specific to the type of disease being treated.

Therapeutic prospects of mesenchymal stem/stromal cells in COVID-19 associated pulmonary diseases:From bench to bedside

The ongoing outbreak of coronavirus disease 2019(COVID-19)caused by the novel severe acute respiratory syndrome coronavirus 2 has become a sudden public emergency of international concern and seriously threatens millions of people’s life health.Two current studies have indicated a favorable role for mesenchymal stem/stromal cells(MSCs)in clinical remission of COVID-19 associated pulmonary diseases,yet the systematical elaboration of the therapeutics and underlying mechanism is far from satisfaction.In the present review,we summarize the therapeutic potential of MSCs in COVID-19 associated pulmonary diseases such as pneumonia induced acute lung injury,acute respiratory distress syndrome,and pulmonary fibrosis.Furthermore,we review the underlying mechanism of MSCs including direct-and trans-differentiation,autocrine and paracrine anti-inflammatory effects,homing,and neovascularization,as well as constitutive microenvironment.Finally,we discuss the prospects and supervision of MSC-based cytotherapy for COVID-19 management before large-scale application in clinical practice.Collectively,this review supplies overwhelming new references for understanding the landscapes of MSCs in the remission of COVID-19 associated pulmonary diseases.

Different priming strategies improve distinct therapeutic capabilities of mesenchymal stromal/stem cells:Potential implications for their clinical use

Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to be safe as a cellular treatment,they have usually been therapeutically ineffective in human diseases.In fact,in many clinical trials it has been shown that MSCs have moderate or poor efficacy.This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs.Recently,specific priming strategies have been used to improve the therapeutic properties of MSCs.In this review,we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs.We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes.Particularly,while hypoxic priming can be used primarily for the treatment of acute diseases,inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders.The shift in approach from regeneration to inflammation implies,in MSCs,a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways.The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential.

Soluble factors secreted by human Wharton’s jelly mesenchymal stromal/stem cells exhibit therapeutic radioprotection: A mechanistic study with integrating network biology

BACKGROUND Human Wharton’s jelly-derived mesenchymal stromal/stem cells(hWJ-MSCs)have gained considerable attention in their applications in cell-based therapy due to several advantages offered by them.Recently,we reported that hWJ-MSCs and their conditioned medium have significant therapeutic radioprotective potential.This finding raised an obvious question to identify unique features of hWJ-MSCs over other sources of stem cells for a better understanding of its radioprotective mechanism.AIM To understand the radioprotective mechanism of soluble factors secreted by hWJMSCs and identification of their unique genes.METHODS Propidium iodide staining,endogenous spleen colony-forming assay,and survival study were carried out for radioprotection studies.Homeostasis-driven proliferation assay was performed for in vivo lymphocyte proliferation.Analysis of RNAseq data was performed to find the unique genes of WJ-MSCs by comparing them with bone marrow mesenchymal stem cells,embryonic stem cells,and human fibroblasts.Gene enrichment analysis and protein-protein interaction network were used for pathway analysis.RESULTS Co-culture of irradiated murine splenic lymphocytes with WJ-MSCs offered significant radioprotection to lymphocytes.WJ-MSC transplantation increased the homeostasis-driven proliferation of the lymphocytes.Neutralization of WJ-MSC conditioned medium with granulocyte-colony stimulating factor antibody abolished therapeutic radioprotection.Transcriptome analysis showed that WJ-MSCs share several common genes with bone marrow MSCs and embryonic stem cells and express high levels of unique genes such as interleukin(IL)1-α,IL1-β,IL-6,CXCL3,CXCL5,CXCL8,CXCL2,CCL2,FLT-1,and IL-33.It was also observed that WJ-MSCs preferentially modulate several cellular pathways and processes that handle the repair and regeneration of damaged tissues compared to stem cells from other sources.Cytokine-based network analysis showed that most of the radiosensitive tissues have a more complex network for the elevated cytokines.CONCLUSION Systemic infusion of WJ-MSC conditioned media will have significant potential for treating accidental radiation exposed victims。

Banking of perinatal mesenchymal stem/stromal cells for stem cellbased personalized medicine over lifetime:Matters arising

Mesenchymal stromal/stem cells(MSCs)are currently applied in regenerative medicine and tissue engineering.Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients.MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices.Usually,clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders.Currently,cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries.Meanwhile,this has led to questions regarding the availability,stability,consistency,multipotency,and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after longterm cryostorage.This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation.This article mainly describes what is known about banking perinatal MSCs in China and,importantly,it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life.This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine,albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.

Long noncoding RNAs in mesenchymal stromal/stem cells osteogenic differentiation:Implications in osteoarthritis pathogenesis

This letter focuses on a recently published article that provided an exceptional description of the effect of epigenetic modifications on gene expression patterns related to skeletal system remodeling.Specifically,it discusses a novel modality of epigenetic regulation,the long noncoding RNAs(lncRNAs),and provides evidence of their involvement in mesenchymal stromal/stem cells osteo-/adipogenic differentiation balance.Despite focus on lncRNAs,there is an emerging cross talk between lncRNAs and miRNAs interaction as a novel mechanism in the regulation of the function of the musculoskeletal system,by controlling bone homeostasis and bone regeneration,as well as the osteogenic differentiation of stem cells.Thus,we touched on some examples to demonstrate this interaction.In addition,we believe there is still much to discover from the effects of lncRNAs on progenitor and non-progenitor cell differentiation.We incorporated data from other published articles to review lncRNAs in normal progenitor cell osteogenic differentiation,determined lncRNAs involved in osteoarthritis pathogenesis in progenitor cells,and provided a review of lncRNAs in non-progenitor cells that are differentially regulated in osteoarthritis.In conclusion,we really enjoyed reading this article and with this information we hope to further our understanding of lncRNAs and mesenchymal stromal/stem cells regulation.

Senescent mesenchymal stem/stromal cells in pre-metastatic bone marrow of untreated advanced breast cancer patients

Breast cancer is the predominant form of carcinoma among women worldwide,with 70%of advanced patients developing bone metastases,with a high mortality rate.In this sense,the bone marrow(BM)mesenchymal stem/stromal cells(MSCs)are critical for BM/bone homeostasis,and failures in their functionality,transform the BM into a premetastatic niche(PMN).We previously found that BM-MSCs from advanced breast cancer patients(BCPs,infiltrative ductal carcinoma,stage III-B)have an abnormal profile.This work aims to study some of the metabolic and molecular mechanisms underlying MSCs shift from a normal to an abnormal profile in this group of patients.A comparative analysis was undertaken,which included self-renewal capacity,morphology,proliferation capacity,cell cycle,reactive oxygen species(ROS)levels,and senescence-associatedβ‑galactosidase(SA‑β‑gal)staining of BMderived MSCs isolated from 14 BCPs and 9 healthy volunteers(HVs).Additionally,the expression and activity of the telomerase subunit TERT,as well as telomere length,were measured.Expression levels of pluripotency,osteogenic,and osteoclastogenic genes(OCT-4,SOX-2,M-CAM,RUNX-2,BMP-2,CCL-2,M-CSF,and IL-6)were also determined.The results showed that MSCs from BCPs had reduced,self-renewal and proliferation capacity.These cells also exhibited inhibited cell cycle progression and phenotypic changes,such as an enlarged and flattened appearance.Additionally,there was an increase in ROS and senescence levels and a decrease in the functional capacity of TERT to preserve telomere length.We also found an increase in pro-inflammatory/pro-osteoclastogenic gene expression and a decrease in pluripotency gene expression.We conclude that these changes could be responsible for the abnormal functional profile that MSCs show in this group of patients.

Harnessing and honing mesenchymal stem/stromal cells for the amelioration of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency.Despite its increased use,the mortality rate for patients undergoing this procedure remains high,mainly due to the perceived risk of exacerbating graft-versushost disease(GVHD).However,even with immunosuppressive agents,some patients still develop GVHD.Advanced mesenchymal stem/stromal cell(MSC)strategies have been proposed to achieve better therapeutic outcomes,given their immunosuppressive potential.However,the efficacy and trial designs have varied among the studies,and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs.This review aims to provide real insights into this clinical entity,emphasizing diagnostic,and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues.The indications and timing for the clinical application of MSCs are still subject to debate.

Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration

Traumatic brain injury is a serious and complex neurological condition that affects millions of people worldwide.Despite significant advancements in the field of medicine,effective treatments for traumatic brain injury remain limited.Recently,extracellular vesicles released from mesenchymal stem/stromal cells have emerged as a promising novel therapy for traumatic brain injury.Extracellular vesicles are small membrane-bound vesicles that are naturally released by cells,including those in the brain,and can be engineered to contain therapeutic cargo,such as anti-inflammatory molecules,growth factors,and microRNAs.When administered intravenously,extra cellular vesicles can cross the blood-brain barrier and deliver their cargos to the site of injury,where they can be taken up by recipient cells and modulate the inflammatory response,promote neuroregeneration,and improve functional outcomes.In preclinical studies,extracellular vesicle-based therapies have shown promising results in promoting recove ry after traumatic brain injury,including reducing neuronal damage,improving cognitive function,and enhancing motor recovery.While further research is needed to establish the safety and efficacy of extra cellular vesicle-based therapies in humans,extra cellular vesicles represent a promising novel approach for the treatment of traumatic brain injury.In this review,we summarize mesenchymal ste m/stromal cell-de rived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration and brainderived extracellular vesicles as potential biofluid biomarkers in small and large animal models of traumatic brain injury.

Adipose stromal/stem cells in regenerative medicine:Potentials and limitations

This article presents the stem and progenitor cells from subcutaneous adipose tissue,briefly comparing them with their bone marrow counterparts,and discussing their potential for use in regenerative medicine.Subcutaneous adipose tissue differs from other mesenchymal stromal/stem cells(MSCs)sources in that it contains a pre-adipocyte population that dwells in the adventitia of robust blood vessels.Pre-adipocytes are present both in the stromal-vascular fraction(SVF;freshly isolated cells)and in the adherent fraction of adipose stromal/stem cells(ASCs;in vitro expanded cells),and have an active role on the chronic inflammation environment established in obesity,likely due their monocyticmacrophage lineage identity.The SVF and ASCs have been explored in cell therapy protocols with relative success,given their paracrine and immunomodulatory effects.Importantly,the widely explored multipotentiality of ASCs has direct application in bone,cartilage and adipose tissue engineering.The aim of this editorial is to reinforce the peculiarities of the stem and progenitor cells from subcutaneous adipose tissue,revealing the spheroids as a recently described biotechnological tool for cell therapy and tissue engineering.Innovative cell culture techniques,in particular 3D scaffold-free cultures such as spheroids,are now available to increase the potential for regeneration and differentiation of mesenchymal lineages.Spheroids are being explored not only as a model for cell differentiation,but also as powerful 3D cell culture tools to maintain the stemness and expand the regenerative and differentiation capacities of mesenchymal cell lineages.

Precise tissue bioengineering and niches of mesenchymal stem cells:Their size and hierarchy matter

Stem cell microterritories(niches),as a specialized part of the extracellular matrix(ECM),are considered an important target and tool for the development of new materials,medical implants,and devices.However,tissue bioengineering products that have stem cell niches of known size on the surface or in the bulk structure of artificial materials are practically unknown.This brief review attempts to draw attention to the problematic aspects of niches as specific parts of the ECM,such as their hierarchy and size for mesenchymal stromal/stem cells(MSCs).These parameters arise directly from numerous definitions of stem cell niches as specialized morphological microterritories found in various tissues.The authors of this review analyze the known information on the hierarchy of MSC microterritories by analogy with that of hematopoietic stem cells.Occasional reports on the size of artificial MSC niches compared to natural niche candidates are summarized.A consensus on a hierarchy and optimal range of niche sizes for MSCs and other stem cells is needed to accelerate the development of prototyping technologies and additive manufacturing in applications to precise tissue bioengineering and regenerative medicine.

Modified mesenchymal stem cells in cancer therapy: A smart weapon requiring upgrades for wider clinical applications

Mesenchymal stem stromal cells(MSC)are characterized by the intriguing capacity to home toward cancer cells after systemic administration.Thus,MSC can be harnessed as targeted delivery vehicles of cytotoxic agents against tumors.In cancer patients,MSC based advanced cellular therapies were shown to be safe but their clinical efficacy was limited.Indeed,the amount of systemically infused MSC actually homing to human cancer masses is insufficient to reduce tumor growth.Moreover,induction of an unequivocal anticancer cytotoxic phenotype in expanded MSC is necessary to achieve significant therapeutic efficacy.Ex vivo cell modifications are,thus,required to improve anti-cancer properties of MSC.MSC based cellular therapy products must be handled in compliance with good manufacturing practice(GMP)guidelines.In the present review we include MSCimproving manipulation approaches that,even though actually tested at preclinical level,could be compatible with GMP guidelines.In particular,we describe possible approaches to improve MSC homing on cancer,including genetic engineering,membrane modification and cytokine priming.Similarly,we discuss appropriate modalities aimed at inducing a marked cytotoxic phenotype in expanded MSC by direct chemotherapeutic drug loading or by genetic methods.In conclusion,we suggest that,to configure MSC as a powerful weapon against cancer,combinations of clinical grade compatible modification protocols that are currently selected,should be introduced in the final product.Highly standardized cancer clinical trials are required to test the efficacy of ameliorated MSC based cell therapies.

Exosomes from adipose tissue-derived stem/stromal cells:A key to future regenerative medicine

Advances in regenerative medicine correlate strongly with progress in the use of adipose tissue-derived mesenchymal stem/stromal cells.The range of therapeutic indications has also expanded over recent years.Numerous recent studies have highlighted the primary importance of paracrine secretion by these cells.Though it is interesting to compare the different types of such secretions,we believe that exosomes(extra-cellular vesicles possessing the same properties as their source cells)will likely be the main key in tomorrow’s cell therapy.Exosomes also have many advantages compared to the direct use of cells,making these particles amajor target in fundamental and translational research.

Effects of storage media,supplements and cryopreservation methods on quality of stem cells

Despite a vast amount of different methods, protocols and cryoprotective agents(CPA), stem cells are often frozen using standard protocols that have beenoptimized for use with cell lines, rather than with stem cells. Relatively fewcomparative studies have been performed to assess the effects of cryopreservationmethods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent forthe development of cryobiology and has been used universally for cryopreservation.However, the use of DMSO has been associated with in vitro and in vivotoxicity and has been shown to affect many cellular processes due to changes inDNA methylation and dysregulation of gene expression. Despite studies showingthat DMSO may affect cell characteristics, DMSO remains the CPA of choice, bothin a research setting and in the clinics. However, numerous alternatives to DMSOhave been shown to hold promise for use as a CPA and include albumin,trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, wewill discuss the use, advantages and disadvantages of these CPAs for cryopreservationof different types of stem cells, including hematopoietic stem cells,mesenchymal stromal/stem cells and induced pluripotent stem cells.

Mesenchymal stem cells in progression and treatment of cancers

Mesenchymal stem or stromal cells （MSCs） from bone marrow or local tissues are recruited to stroma of almost all types of cancers during initiation and/or progression of cancer. The recruited MSCs and their derivative cancer-associated fibroblasts interact with cancer cells to promote sternness, invasion and metastasis of cancer cells. Targeting these cancer-recruited MSCs and/or the interaction between MSCs and cancer cells are promising strategies to improve cancer therapy. On the other hand, the unique tumor-homing capacity of MSCs makes them a promising vehicle to deliver various anti-cancer agents. This review summarized the recent advancement of our understanding on the interaction between MSCs and cancer ceils, as well as the potential of MSCs for cancer therapy.

NAD^(+) salvage governs the immunosuppressive capacity of mesenchymal stem cells

Mesenchymal stem/stromal cells(MSCs)possess robust immunoregulatory functions and are promising therapeutics for inflammatory disorders.This capacity is not innate but is activated or‘licensed’by inflammatory cytokines.The licensing mechanism remains unclear.Here,we examined whether inflammatory cytokines metabolically reprogrammed MSCs to confer this immunoregulatory capacity.In response to stimulation by inflammatory cytokines,MSCs exhibited a dramatic increase in the consumption of glucose,which was accompanied by an enhanced use of nicotinamide adenine dinucleotide(NAD^(+))and increased expression of nicotinamide phosphoribosyltransferase(NAMPT),a central enzyme in the salvage pathway for NAD^(+) production.When NAD^(+) synthesis was blocked by inhibiting or depleting NAMPT,the immunosuppressive function of MSCs induced by inflammatory cytokines was greatly attenuated.Consequently,when NAD^(+) metabolism in MSCs was perturbed,their therapeutic benefit was decreased in mice suffering from inflammatory bowel disease and acute liver injury.Further analysis revealed that NAMPT-driven production of NAD^(+) was critical for the inflammatory cytokine-induced increase in glycolysis in MSCs.Furthermore,the increase in glycolysis led to succinate accumulation in the tricarboxylic acid cycle,which led to hypoxia-inducible factor 1α(HIF-1α)stabilization and subsequently increased the transcription of key glycolytic genes,thereby persistently maintaining glycolytic flux.This study demonstrated that unlike its proinflammatory role in immune cells,NAD^(+) metabolism governs the anti-inflammatory function of MSCs during inflammation.

Stem cell-derived hepatocyte therapy using versatile biomimetic nanozyme incorporated nanofiber-reinforced decellularized extracellular matrix hydrogels for the treatment of acute liver failure

Reactive oxygen species(ROS)-associated oxidative stress,inflammation storm,and massive hepatocyte necrosis are the typical manifestations of acute liver failure(ALF),therefore specific therapeutic interventions are essential for the devastating disease.Here,we developed a platform consisting of versatile biomimetic copper oxide nanozymes(Cu NZs)-loaded PLGA nanofibers(Cu NZs@PLGA nanofibers)and decellularized extracellular matrix(dECM)hydrogels for delivery of human adipose-derived mesenchymal stem/stromal cells-derived hepatocyte-like cells(hADMSCs-derived HLCs)(HLCs/Cu NZs@fiber/dECM).Cu NZs@PLGA nanofibers could conspicuously scavenge excessive ROS at the early stage of ALF,and reduce the massive accumulation of pro-inflammatory cytokines,herein efficiently preventing the deterioration of hepatocytes necrosis.Moreover,Cu NZs@PLGA nanofibers also exhibited a cytoprotection effect on the transplanted HLCs.Meanwhile,HLCs with hepatic-specific biofunctions and anti-inflammatory activity acted as a promising alternative cell source for ALF therapy.The dECM hydrogels further provided the desirable 3D environment and favorably improved the hepatic functions of HLCs.In addition,the pro-angiogenesis activity of Cu NZs@PLGA nanofibers also facilitated the integration of the whole implant with the host liver.Hence,HLCs/Cu NZs@fiber/dECM performed excellent synergistic therapeutic efficacy on ALF mice.This strategy using Cu NZs@PLGA nanofiber-reinforced dECM hydrogels for HLCs in situ delivery is a promising approach for ALF therapy and shows great potential for clinical translation.

Local administration of porcine immunomodulatory,chemotactic and angiogenic extracellular vesicles using engineered cardiac scaffolds for myocardial infarction

The administration of extracellular vesicles(EV)from mesenchymal stromal cells(MSC)is a promising cell-free nanotherapy for tissue repair after myocardial infarction(MI).However,the optimal EV delivery strategy remains undetermined.Here,we designed a novel MSC-EV delivery,using 3D scaffolds engineered from decellularised cardiac tissue as a cell-free product for cardiac repair.EV from porcine cardiac adipose tissue-derived MSC(cATMSC)were purified by size exclusion chromatography(SEC),functionally analysed and loaded to scaffolds.cATMSC-EV markedly reduced polyclonal proliferation and pro-inflammatory cytokines production(IFNγ,TNFα,IL12p40)of allogeneic PBMC.Moreover,cATMSC-EV recruited outgrowth endothelial cells(OEC)and allogeneic MSC,and promoted angiogenesis.Fluorescently labelled cATMSC-EV were mixed with peptide hydrogel,and were successfully retained in decellularised scaffolds.Then,cATMSC-EV-embedded pericardial scaffolds were administered in vivo over the ischemic myocardium in a pig model of MI.Six days from implantation,the engineered scaffold efficiently integrated into the post-infarcted myocardium.cATMSC-EV were detected within the construct and MI core,and promoted an increase in vascular density and reduction in macrophage and T cell infiltration within the damaged myocardium.The confined administration of multifunctional MSC-EV within an engineered pericardial scaffold ensures local EV dosage and release,and generates a vascularised bioactive niche for cell recruitment,engraftment and modulation of short-term post-ischemic inflammation.