Advances in mesenchymal stem cell therapy for immune and inflammatory diseases: Use of cell‐free products and human pluripotent stem cell‐derived mesenchymal stem cells

Abstract Mesenchymal stem cell therapy (MSCT) for immune and inflammatory diseases continues to be popular based on progressive accumulation of preclinical mechanistic evidence. This has led to further expansion in clinical indications from graft rejection, autoimmune diseases, and osteoarthritis, to inflammatory liver and pulmonary diseases including COVID‐19. A clear trend is the shift from using autologous to allogeneic MSCs, which can be immediately available as off‐the‐shelf products. In addition, new products such as cell‐free exosomes and human pluripotent stem cell (hPSC)‐derived MSCs are exciting developments to further prevalent use. Increasing numbers of trials have now published results in which safety of MSCT has been largely demonstrated. While reports of therapeutic endpoints are still emerging, efficacy can be seen for specific indications—including graft‐vs‐host‐disease, strongly Th17‐mediated autoimmune diseases, and osteoarthritis—which are more robustly supported by mechanistic preclinical evidence. In this review, we update and discuss outcomes in current MSCT clinical trials for immune and inflammatory disease, as well as new innovation and emerging trends in the field.


| INTRODUCTION
Mesenchymal stem/stromal cells (MSCs) are multipotent progenitor cells capable of supporting hematopoiesis and differentiation into the multiple mesodermal lineages of osteoblasts, chondrocytes, and adipocytes. [1][2][3] First found in the bone marrow (BM), MSCs have been isolated from numerous organs/tissues over the past several decades, but in vivo identity remains somewhat elusive, with increasing evidence for a perivascular origin. [4][5][6] An unexpected function of MSCs-especially prominent with human sources-is its strong immunomodulatory properties, which have been best delineated toward CD4 cells but also well characterized against a variety of myeloid and innate leukocytes, including dendritic cells, monocytes, and macrophages. [7][8][9] While initial preclinical data of MSC therapeutic efficacy were mainly focused on regenerative and differentiation capacity, it quickly became apparent that the immunomodulatory properties not only are clinically relevant but allow for allogeneic, unmatched use of these progenitor cells. The application of MSCs toward immune and inflammatory diseases rapidly ensued, with a doubling of clinical trials for these conditions within the past 5 years. 10 Moreover, discoveries of new mechanisms and new products-including using MSC-derived products and human pluripotent stem cell (hPSC) including embryonic stem cell (ESC) and induced PSC (iPSC)-derived MSCs-as well as emerging diseases such as COVID-19 has continued to widen the clinical application of MSC immunomodulation. 11 We therefore review the current status of clinical trials using MSC therapy (MSCT) for inflammatory or immune-related diseases and discuss new advances in the field.

| BRIEF SUMMARY ON PRECLINICAL EVIDENCE OF MSC IMMUNOMODULATORY MECHANISMS
The immunomodulatory properties of MSCs are well demonstrated toward both lymphoid and myeloid cells, with increasing accumulation of mechanistic evidence (Figure 1). MSC immune functions have been best documented against CD4 T lymphocytes, a critical leukocyte population in orchestrating overall immune responses. Numerous reports have shown that MSCs modulate these adaptive cells from an inflammatory milieu filled predominantly with effector T cells to a regulatory T (Treg)-rich microenvironment largely through paracrine factors, most commonly through transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF) 12 prostaglandin E 2 (PGE 2 ), 13 nitric oxide (NO), and indoleamine 2,3-dioxygenase (IDO). 8,[14][15][16] While a few studies found cell-cell contact involved in MSC-T cell immunomodulation, 17 this mechanism is more prominent in MSC-NK interactions, involving downregulation of activating NK receptors F I G U R E 1 Mechanisms of mesenchymal stem cell (MSC) immunomodulation toward lymphoid and myeloid cells as evidenced in preclinical in vivo studies. NK, natural killer cell; PMN, polymorphonuclear neutrophil; MΦ, macrophage; MDSC, myeloid-derived suppressor cell; DC, dendritic cell; PGE 2 , prostaglandin E2; IDO, Indoleamine 2,3-dioxygenase; HLA-G, human leukocyte antigen-G; TGF-β, transforming growth factor beta; Gal, galectin; IL, Interleukin; NO, nitric oxide; HO-1, heme oxygenase-1; HGF, hepatocyte growth factor; PD-L, programmed death-ligand; IL-1RA, interleukin-1 receptor antagonist; CCL2, chemokine ligand 2; TSG-6, TNF-stimulated gene 6 protein; SOD3, superoxide dismutase 3; SCT-1, stanniocalcin-1; IGF-1, insulin-like growth factor-1; KGF, keratinocyte growth factor; GRO-γ, growth related oncogene γ. Cell-contact factors are denoted in brackets such as KIR, NKp30, NKp44 and NKG2D through MSC-expressed   surface and soluble HLA-G, a non-classical MHC class I molecule   important in fetal-maternal immunomodulation. 18,19 Interestingly,   immunomodulatory paracrine factors such as PGE 2 and IDO inducible   by inflammatory signals including IFN-γ and IL-1β are prominent in   MSC interactions across leukocyte subpopulations including all lymphoid   cells including T cells, NKs, 20 and B cells in which IL-10-expressing regulatory B cells are expanded. 21,22 Reports are most scarce for MSC-B cell interactions, but most demonstrate suppression of B cell proliferation, differentiation, and antibody production [23][24][25][26][27][28][29] ; our recent report found that MSC-B cell interactions may be more complex than previously thought due to MSC source-specific differences in expression of relevant factors. 30 Such information on tissue-specific MSC properties may provide insights which could prove relevant for clinical application. 11,31 The broad reach of MSC immunomodulation is best exemplified by interactions with myeloid cells, which are much more heterogenous than lymphoid cells. Among early reports of MSC modulation are studies on dendritic cells (DCs)-professional antigen-presenting cells that initiate T cell response-in which MSC paracrine factors including IL-6, 32 PGE 2 , 33 and growth-regulated oncogene (GRO)-γ, 34 as well as cell-cell contact through Jagged-2 35 , suppress maturation and lead to the development of regulatory DCs, which are more immature and tolerogenic. MSCs have also been seen to inhibit activation of macrophages and/or induce polarization into an alternative M2 phenotype which are critical in resolving inflammation, with data indicating involvement of paracrine factors including IL-1RA, 36 45 and multiple anti-bacterial functions of PMNs through IL-1β secretion. 48 Such accumulation and broadening of evidence for MSC interactions with numerous leukocyte populations is extremely relevant for better tailoring of MSCT-that is, determine which tissuespecific sources or cell-derived products to use-for more effective targeting of specific diseases and/or patient subpopulations.  Table 1). Only a few trials are in Phase 3 to determine effectiveness (n = 18 or 3.7%), combined Phase 2/3 studies  remarkably, there is a very recent trial using iPSC-MSCs (n = 1). However, 28 trials did not specify the source of MSCs, and two used MSC-derived products with one using exosomes from WJUCMSCs and one using conditioned medium (CM) from unspecified MSCs.  Notes: a Trial using two sources of MSCs: WJUC and amnion; b,c,d,e,f Trials using two sources of MSCs: BM and WJUC; g Trial using two sources of MSCs: BM and placenta; i Trial using two sources of MSCs: BM and adipose; h Trial using two sources of MSCs: BM and cord blood; j,k,l Trials using both the MSC and its derived products. A Reprogrammed from peripheral blood mononuclear cells. B Exosomes or trophic factors collected from conditioned medium.
Interestingly, the majority of trials use allogeneic rather than autologous MSCs (57 vs 16 trials, respectively), while one trial used both types and 19 trials were undefined.
Because graft rejection was the first clinical application of MSCT, this field has the most published studies of clinical data so far, with nine reports on GVHD and 11 reports on solid organ transplantation.
Several adult and pediatric trials-including two Phase 3 trials-for steroid-refractory acute or chronic GVHD using intravenous infusion of allogenic BMMSCs at doses of 1$2 Â 10 6 /kg showed significant therapeutic efficacy and safety [63][64][65][66][67][68] ; moreover, co-transplantation of BMMSCs at the time of HSCT was found to prevent GVHD progression and/or occurrence. 69,70 One of the most exciting results is a Phase 1 trial published this year using allogeneic iPSC-MSCs for steroid-resistant acute GVHD, in which safety and some efficacy was seen. 71 Interestingly, for solid organ rejection, all seven published reports used autologous BMMSCs for renal transplantation, 72-77 and pancreatic islet cell transplantation. 78 Immunosuppressive drugs were used in all these studies except in the islet cell transplantation study and all demonstrated safety, with some efficacy seen in these studies.
Allogeneic BMMSCs or WJUCMSC were used in two studies of renal transplantation, and no toxicity was seen. 79,80 When considering the efficacy of allogenic MSCs, WJUCMSCs was reported to successfully prevent both delayed graft function and acute rejection in renal transplantation, 81 whereas BMMSCs were not found to induce immunosuppression in a report on liver transplantation. 82 These published results overall demonstrate that for graft rejection, MSCT is safe and may be efficacious, especially for pediatrics cases of GVHD where allogeneic BMMSC therapy may be particularly beneficial.

| Autoimmune diseases
Autoimmune diseases are disorders in which the body's immune system attacks its own cells and organs, and autoreactive T  WJUCMSCs to CD and ulcerative colitis (UC) patients also reduced mucosal inflammation. 87,88 The six published reports using autologous BMMSCs administered intravenously or intrathecally for MS demonstrated safety and some non-significant reduction of inflammatory parameters [89][90][91][92][93][94] ; two other reports utilized allogeneic WJUCMSCs, with one study still ongoing 95

| Pulmonary inflammation and COVID-19
The high entrapment of cells within the lungs after intravenous injection-the most common method to deliver cell therapy-has long been known, and can be taken advantage of in MSCT for pulmonary diseases. 134 As an organ open to the environment, a number of infec- allogeneic BMMSCs. 148 Clinical results of MSCT for lung diseasesespecially COVID-19-are highly anticipated because of the invaluable information these numerous trials will provide on the efficacy of not only MSCT but also MSC-related products.

| Liver cirrhosis
Liver fibrosis or cirrhosis is the end-stage manifestation of many hepatic diseases, ranging from infectious insults due to hepatitis viruses B and C, to non-infectious conditions such as alcohol abuse and non-alcoholic fatty liver disease; it is also a risk factor for hepatocellular carcinoma formation. 149 Compared with other organ systems,  156 We also recently demonstrated that PMSC treatment in a mouse model of hypervirulent Klebsiella-induced severe intra-abdominal infection can enhance neutrophil bactericidality to reduce liver injury and increase survival. 48 In a mouse model of autoimmune cholangitis, liver inflammation was reduced through WJUCMSC-secreted Gal-9. 157 These increasing numbers of preclinical reports support that MSC immunomodulation may be efficacious toward hepatitis and cirrhosis.
Currently, there are 40 registered trials of MSCT for liver failure involving viral hepatitis or cirrhosis, with trials predominantly in early phase: six trials in Phase 1, 12 in Phase 2, and 18 in combined Phase1/2 (Table S5) showed limited improvement on short-term outcome of HBV-related liver failure patients. 159 Further accumulation of clinical data is urgently needed to assess whether MSCT is efficacious for inflammation-mediated liver failure and/or cirrhosis.

| NEW DEVELOPMENTS: USE OF MSC-DERIVED PRODUCTS AND HPSC-MSCS FOR IMMUNE-RELATED DISEASES
While the minimal reports of adverse events so far in the large number of MSC clinical trials are reassuring, efficacy has been as easy to achieve as was expected. This has increasingly led to the idea that MSC immunomodulation may be represent short-term immune evasion rather than long-term immune privilege, and that the MSC itself likely rapidly undergo apoptosis after administration. 160 165 macrophages, 166 and DCs. 167 To date, there are 16 trials using MSC-derived products including CM, EVs, or exosomes: three trials are in Phase 1, two in Phase 2, seven are combined Phase 1/2, two are combined Phase 2/3 trials, and two did not specify phase (Table 2). Interestingly, most of the trials (n = 10) are for pulmonary diseases, 11 (Table S6). It can be anticipated that clinical trials using MSC-derived products-readily available as off-the-shelf products-will continue to increase.
The ability to isolate MSCs from a myriad of organs/tissues does not solve the problem that all tissue-derived MSCs undergo senescence, which not only decreases proliferative capacity but differentiation capacity as well. 168 These concerns can be precluded with stringent QA/QC protocols to exclude undifferentiated and genetically unstable cells. 177 In addition, the functional variability with primary-isolated human samples and cell products is avoided since MSCs can be generated from a particular ESC or iPSC line essentially indefinitely to continually provide functionally stable lots of cells and cell products. 178  milestone. 71 Based on increasing preclinical data and these four sentinel trials, it is anticipated that more clinical trials using hPSC-derived MSCs will be conducted.

| PERSPECTIVES ON CHALLENGES IN MSCT
Although there are over 1000 trials using various types of MSCs or MSC-free derivatives being conducted in more than 40 countries, only nine MSC-based products have been approved worldwide for either regenerative or immune-related diseases. 187 Encouragingly, these approved MSC-based products overwhelmingly except for one prod- While clinical effectiveness is ultimately the criteria for approval of therapeutic products, the low numbers of approved MSC products also reflect large national/regional differences in regulation for cellbased products, 188 as well as difficulties in transitioning from preclinical to clinical platforms. 189 MSC products in particular suffers from a lack of agreement on robust and relevant characterization criteria for clinical reliability and functionality; the current Minimal Criteria for MSCs date back nearly two decades and was not established for clinical use nor take into account immunomodulatory properties which were largely discovered after these criteria were agreed upon.
Moreover, "gold standard" double-blind randomized clinical trialincluding testing for critical parameters such as dose, delivery route, and timing-are clearly more difficult to conduct with complex and live cell-based products than pharmaceutical products. 178 Continually progress to overcome these hurdles is occurring which should allow for clinical effectiveness to be more evident in the near future. 187   GVHD, predominately Th17-mediated autoimmune diseases such as IBD and MS, and OA in which both the regenerative and immunomodulatory capacity of MSCs can be useful. New developments in use of cell-free products and iPSC-MSCs, as well as more preclinical data on tissue-specific differences in MSC sources are all likely to further improve MSCT outcomes in the very near future.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.