Stem cells in the treatment of diabetes mellitus

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Corresponding authorGünter Päth, PhDDivision of Endocrinology and DiabetologyDepartment of Medicine IIMedical Center – University of FreiburgFaculty of Medicine, University of FreiburgHugstetter Str. 5579106 FreiburgGERMANYTel.: +49 761 270 73270Fax: +49 761 270 33720E-mail: guenter.paeth@uniklinik-freiburg.deKeywordsStem cells, diabetes mellitus, transplantation, embryonic stem cells, induced pluripotent stemcells, mesenchymal stromal cellsACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT2AbbreviationsADA, American Diabetes Association; AE, adverse event; α-SMA, alpha smooth muscleactin; ANXA1, annexin-1; AUC, area under the curve; BAD, Bcl-2 antagonist of cell death;Bcl-2b, B-cell lymphoma 2; bFGF, basic fibroblast growth factor; BM, bone marrow; BMI,body mass index; CAR cell, CXCL12-abundant reticular cell; CCL5/RANTES, CCchemokineligand 5; CD, cluster of differentiation; CFU-F, colony forming unit-fibroblast;CXCL12, CXC chemokine ligand 12; DC, dendritic cell; Ebf, early B-cell factor; ECM,extracellular matrix; EMT, epithelial to mesenchymal transition; ERK, extracellular signalregulatedkinases; ESC, embryonic stem cells; FDA, Food and Drug Administration; FGFR1,fibroblast growth factor receptor 1; GAD, glutamic acid decarboxylase; GCV, ganciclovir;GM-CSF, granulocyte-macrophage colony-stimulating factor; GSIS, glucose-stimulatedinsulin secretion; GVHD, graft-versus-host disease; HbA1c, glycated hemoglobin A1c; HGF,hepatocyte growth factor; HLA, human leukocyte antigen; HNF1A, hepatocyte nuclear factor1α; HOMA, homeostasis model assessment; HSC, hematopoietic stem cells; HSV-Tk, herpessimplex virus thymidine kinase; ICAM1, intercellular adhesion molecule 1; ICOS, induciblecostimulator; IDDM, insulin-dependent diabetes mellitus; IDO, indoleamine 2,3-dioxygnase;IGF, insulin-like growth factor; IL, interleukin; IL-1RA, interleukin-1 receptor antagonist;IPF-1, insulin promoter factor 1 (official name, PDX1); IR, insulin resistance; iPSC, inducedpluripotent stem cells; ISC, insulin screting cell; ISL-1, insulin gene enhancer protein 1; IU,international unit; IV, intravenous; MET, mesenchymal to epithelial transition; MHC, majorhistocompatibility complex; MMP, matrix metalloproteases; MMTT, mixed-meal tolerancetest; MNC, mononuclear cells; MODY3, maturity-onset diabetes of the young type 3; MPC,mesenchymal precursor cells; MSC, mesenchymal stem cells; NG2, neural/glial antigen 2;NGF, nerve growth factor; NGN3 neurogenin 3; NKX6.1, NK6 homeobox 1; NO, nitricoxide; NOD, non-obese diabetic; NSG, NOD scid gamma; PAX, paired box protein; PDGF,platelet-derived growth factor; PD-L1, programmed cell death ligand 1; PDX1, pancreatic andduodenal homeobox 1; PEDF, pigment epithelium-derived factor; PGE2, Prostaglandin E2;SC, stem cells; SCF, stem cell factor; SCID, severe combined immunodeficiency; STZ,streptozotocin; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; TGF,transforming growth factor; Th, T helper; TNF, tumour necrosis factor; TRAIL, TNF-relatedapoptosis inducing ligand; Treg, regulatory T cell; TSG-6, tumour necrosis factor-stimulatedgene 6; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling;UC, umbilical cord; VCAM1, vascular cell adhesion molecule 1; VEGF, vascular endothelialgrowth factor; WHO, world health organisation; WJ, Wharton's Jelly.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT3AbstractDiabetes mellitus type 1 and type 2 have become a global epidemic with dramaticallyincreasing incidences. Poorly controlled diabetes is associated with severe life-threateningcomplications. Beside traditional treatment with insulin and oral anti-diabetic drugs, clinicianstry to improve patient's care by cell therapies using embryonic stem cells (ESC), inducedpluripotent stem cells (iPSC) and adult mesenchymal stem cells (MSC). ESC display avirtually unlimited plasticity, including the differentiation into insulin producing β-cells, butthey raise ethical concerns and bear, like iPSC, the risk of tumours. IPSC may further inheritsomatic mutations and remaining somatic transcriptional memory upon incomplete reprogramming,but allow the generation of patient/disease-specific cell lines. MSC avoid suchissues but have not been successfully differentiated into β-cells. Instead, MSC and theirpericyte phenotypes outside the bone marrow have been recognized to secrete numerousimmunomodulatory and tissue regenerative factors. On this account, the term 'medicinalsignaling cells' has been proposed to define the new conception of a 'drug store' for injuredtissues and to stay with the MSC nomenclature. This review presents the biologicalbackground and the resulting clinical potential and limitations of ESC, iPSC and MSC, andsummarizes the current status quo of cell therapeutic concepts and trials.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT4IntroductionHuman organs and tissues possess a limited capacity to completely recover their structure andfunction in a number of pathologic conditions and degenerative diseases. This fact initiatedthe multidisciplinary field of regenerative medicine which investigates the potential of stemcells (SC) for tissue repair and restoration of organ function. Based on their nature and origin,SC exhibit features of interest for cell therapies; e.g. targeting functional degeneration andloss of insulin producing pancreatic β-cells in diabetes mellitus (DM). The diverse potential ofembryonic SC (ESC), induced pluripotent SC (iPSC) and adult mesenchymal SC (MSC) hasbeen exploited to restore or maintain insulin secretion as well as to investigate patient-specificdisease aspects. MSC are currently the most investigated cells in DM-related trials whileclinical testing of ESC has just started. This review summarizes the biological aspects and theapplication strategies for the treatment of DM by stem cell therapy.β-cell replacementPatients with autoimmune DM type 1 (T1DM) experience a loss of insulin producingpancreatic β-cells and rely on daily insulin injections. Despite modern insulin therapies,exogenous application of insulin can never be as accurate and dynamic like insulin secretionfrom endogenous β-cells and therefore can only partially reduce the risk for the developmentof micro- (i.e. nephropathy, retinopathy) or macrovascular (i.e. coronary artery disease,peripheral artery disease, cerebrovascular disease) complications. Additionally, efforts todevelop effective immunosuppressive treatments to prevent β-cell loss before disease onsethad limited success so far [1]. Consequently, restoration of endogenous insulin secretionrepresents an important aim to prevent hyper- and hypoglycemia as well as to reduce or avoiddiabetic complications and the patient's requirement for self-management of glycemic controlby exogenous insulin administration.Clinical islet transplantation aims to re-establish endogenous insulin secretion and has beensteadily refined since its beginning in the 1980s [2]. An important step was the 'EdmontonProtocol' from 1999 which avoids β-cell toxic glucocorticoids by using sirolimus, tacrolimusand daclizumab for immunosuppression [3]. Ongoing clinical research improved isolation,culture and transplant techniques, and evaluated advanced anti-inflammatory andACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT5immunomodulatory interventions [4-6]. As a result, a multicenter analysis with 18 diabeticpatients receiving 34 islet transplantations showed a graft survival (defined by C-peptideconcentrations of ≥ 0.3 ng/ml) of about 72.2%, 44.4% and 22.2% after 1, 2 and 5 years,respectively [7]. Subsequently, a multicenter phase 3 trial, which enrolled 48 participantsreceiving 75 islet transplantations, successfully improved glycemic control to a medianglycated hemoglobin A1c (HbA1c) level of 5.6% after 1 and 2 years [8]. Compared tostandard insulin therapy, islet transplantation more efficiently improved glycemic control andprogression of retinopathy, and resolved hypoglycemia even in patients with only partiallyremaining graft function [9, 10].Transplantation of whole pancreases is an established alternative to islets and both proceduresdisplay advantages and limitations [11]. The standard procedure of islet infusion into the liveris much safer with less complications than pancreas transplantation which is considered amajor surgery with accordingly enhanced risks for the patient. Thus, pancreas transplantationis rarely performed alone and is most commonly combined with a kidney transplantation inpatients with T1DM and end-stage renal disease. The major obstacle of the less risky islettransplantation is the limited graft survival. Insulin independence after islet transplantationinitially reached barely 10% at 1 year but has been improved by the Edmonton protocol to 10-15% at 5 years [12] and by later developments, such as T cell-depleting agents and blockadeof tumour necrosis factor (TNF), to 50% at 5 years [4]. Thereby, at least experienced islettransplantation centers have substantially improved long-term graft function towards thereported 5 year outcomes in pancreas transplantation; 55% for pancreas alone and afterkidney, and 72% for pancreas together with kidney [2, 13].As with other organ transplantation, there is a great scarcity of human donor material whichevoked intensive research on the generation of insulin producing β-cells from SC. Wholepancreas transplantation strictly depends on high quality organs while generation of insulinproducing β-cells from SC has the potential to solve the problem of limited availability ofdonor material for islet transplantation. However, the need for immunosuppression reservesboth pancreas and islet transplantation as a therapeutic choice to a limited patient populationsuch as brittle diabetics with life-threatening hypoglycemic events or subjects who anywayreceive immunosuppression; e.g. because of kidney transplantations for renal failure due todiabetic nephropathy.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT6I. ESCESC represent the inner cell mass of the blastocyst and possess a pluripotent differentiationcapacity. This makes them capable to form all three germ layers (ectoderm, endoderm andmesoderm) which subsequently give rise to all cell types of the body. On this account, thesecells are considered the superior tool for tissue generation but they evoke ethical concernsregarding their origin from human embryos. Moreover, they bear a clinical risk since theirpluripotent nature makes undifferentiated ESC capable to form teratomas and malignantteratocarcinomas in vivo. The development of clinically safe differentiation protocols andtesting routines for tumorigenicity in rodents represents an important challenge in the field[14, 15].The differentiation of human ESC into functional β-cells is not trivial since transformingprocesses have to mimic complex embryonal organogenesis in vitro. Differentiation protocolstherefore established a number of factors and inhibitors that modulate molecular pathways inan exact sequential timing to resemble the natural development of pancreatic β-cells.Generally, human ESC were firstly differentiated into definitive endoderm cells and thensequentially into primitive gut tube and posterior foregut, pancreatic endoderm and finally β-cells using multiple specified media and supplementation in each step [16, 17]. The earlierattempts proved the concept but did not achieve high yields; e.g. a typical early study resultedin an average percentage of insulin+ cells in differentiated human ESC cultures of 7.3% [16].Meanwhile, the exact media compositions have been further improved and current protocolsreached the level of scalable production of β-cell phenotypes with functional insulin secretion[17-20]. However, the complexity of protocols and differences in employed ESC and iPSClines raise difficulties in reproducibility outside experienced laboratories since stem celldifferentiation is despite all progress not a routine procedure.A major step in differentiation of human ESC towards β-cells is the expression of thetranscription factors pancreatic and duodenal homeobox 1 (PDX1) and NK6 homeobox 1(NKX6.1) which are markers of pancreatic endoderm and endocrine precursor cells. It hasbeen shown that comparable human embryonic pancreas tissue from fetal weeks 6-9, whichcontained very few β-cells at that stage, is capable to mature into functional β-cells aftertransplantation into non-obese diabetic mice with severe combined immunodeficiency(NOD/SCID mice) [21]. Based on these findings, in vivo maturation of PDX1+/NKX6.1+ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT7progenitors into β-cells has been recognized to be more efficient than mimicking this monthslongprocess in vitro [22]. Furthermore, scalable in vitro differentiation of ESC into endocrinepancreatic precursor cells is to date more robust and less complicated [23, 24] than generationof fully functional β-cell phenotypes by advanced protocols [17, 19] and therefore thefavoured strategy of the company ViaCyte [25].The resulting improvements in differentiation of ESC towards PDX1+/NKX6.1+ pancreaticprogenitors during the last decade have also reduced the risk of tumour formation in vivowhich occurred in a typically early study of the company NovoCell (name changed toViaCyte in 2010) with a rate of 7 out of 46 transplanted mice [22]. Since then, ViaCyte hassteadily optimized the approach to efficient large-scale production and embedded theirpancreatic endoderm and endocrine progenitor cells into a macroencapsulation device togenerate immune isolated VC-01 implants [18, 25, 26].Macroencapsulation improved graft survival and clinical safety. Although ESC-derivedprogenitors are hypoimmunogenic, transplanted cells are challenged by adaptive immuneresponses such as local inflammation and rejection. In addition, once maturated into insulinproducing β-cells, graft cells will be attacked by persistent autoreactive T cells in patientswith T1DM. Several studies have demonstrated that macroencapsulation protects embeddedcells by isolation from immune responses and thereby avoids rejection and the need forimmunosuppression [27, 28]. Furthermore, macroencapsulation prevents escape of embeddedcells into the body. This is an important safety issue since any ESC transplanted in anundifferentiated state bears the potential of malignant transformation. In this view,subcutaneously transplanted devices could be retrieved and removed easily. Using thisconcept, Viacyte has achieved a milestone by initiating the first-in-man clinical trial to testsafety and efficacy of their pancreatic endoderm implant VC-01 in patients with T1DM andhypoglycemic unawareness (ClinicalTrials.gov: NCT02239354, currently enrolling patients,estimated completion in January 2021).This trial will answer the question whether the established in vivo maturation of human ESCderivedPDX1+/NKX6.1+ progenitors in rodents can be comparably recapitulated in humansubjects. In addition, two of three further trials initiated by Viacyte aim to test a modifiedimplantation device with reduced immuno isolation that allows vascularisation of themacroencapsulated cells. Provided that outcome of all these trials will prove safety, efficacyACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT8and significant long-term graft function, this approach holds the potential to pave the waytowards clinical β-cell replacement without immunosuppression and independent of thelimited availability of donor islets.II. iPSCThe ethical criticism related to the use of human pre-implantation embryos for extraction ofESC inspired researchers to refine the work of John B. Gurdon. His key study demonstratedthat enucleated Xenopus laevis eggs that were transplanted with nuclei from differentiatedintestinal epithelium could, at least in small numbers, develop into living tadpoles [29]. Thisdemonstrated for the first time that somatic cell nuclei have the potential to revert into apluripotent state. Decades passed by before this finding gained broad acceptance in thescientific community and the progression of this idea culminated in the birth of the firstmammal, clone sheep Dolly, on July 5, 1996 [30].Gurdons basic concept of reprogrammable somatic cells was further developed and onedecade after Dolly's birth, Takahashi and Yamanaka induced embryonic-like pluripotency insomatic mouse fibroblasts by viral overexpression of the four transcription factors Sox2,Oct4, Klf4 and c-Myc [31]. For their groundbreaking discoveries Gurdon and Yamanaka werehonored by the Noble Prize in 2012 (www.nobelprize.org).As a major achievement, iPSC overcome the ethic obstacle of using embryos for harvest ofESC. During ongoing research, the original Yamanaka protocol has been diversely modifiedand viral integration was effectively replaced by treatment with recombinant proteins, smallmolecules and microRNAs [32, 33]. Following the principle route of pancreatic developmentas used for ESC, researchers have successfully differentiated iPSC into functional β-cellphenotypes [34, 35] and also established scalable production of both endocrine pancreaticprogenitors and β-cells [20, 36].Nevertheless, further refinement of procedures is still an issue and new tools were created forthe identification of compounds and conditions which enhance yield and functionality ofgenerated β-cells. For example, human iPSC expressing the fluorescent reporters Venus andmCherry markers under the control of intrinsic neurogenin 3 and insulin promoters have beenACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT9generated for screening of differentiation efficiency [37]. These cells have served to identifyan inhibitor of fibroblast growth factor receptor 1 (FGFR1) that, while blocking the earlydevelopment of pancreatic progenitors, promoted the terminal differentiation of pancreaticendocrine progenitors into endocrine cells including β-cells.However, due to their origin from adult somatic cells, iPSC can inherit somatic mutations andincomplete reprogramming can maintain somatic transcriptional memory including cancerassociated gene activity [38, 39]. These dangers currently do not define them as the firstchoice for clinical use but, more importantly, iPSC enable the successful generation ofpatient-specific cell phenotypes that allow to recapitulate disease processes in vitro and canserve as platforms for drug development and testing [40-42]. For example, researcherssuccessfully generated an iPSC line from a patient carrying a hepatocyte nuclear factor 1α(HNF1A) mutation resulting in maturity-onset diabetes of the young type 3 (MODY3). In thenear future, patient-specific cell lines will help to develop disease-related models thatovercome the obstacle of species differences between human subjects and animal models.III. MSCFor an excellent graphical overview on MSC biology discussed in section III we recommendthe poster by Somoza et al. [43].MSC within the bone marrow (BM)The discovery of MSC has been generally attributed to A. J. Friedenstein who observed thatBM explants form plastic adherent fibroblast-like clonogenic cells with a high replicativecapacity in vitro and named them colony forming unit-fibroblasts (CFU-F) [44]. Friedensteinet al. further figured out that culture expanded CFU-F are capable to differentiate intoosteoblasts, chondrocytes and adipocytes, and to reconstitute a hematopoieticmicroenvironment after transplantation in irradiated mice [45, 46]. These findings supportedthe pioneering study of Tavassoli and Crosby who demonstrated that autologous BMfragments transplanted into extramedullary sites can reconstitute hematopoietic andadventitial structures in rats [47]. The observed process started from a developing network ofproliferating reticular cells and was successively followed by the occurrence of osteoblasts,ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT10osteoid tissue, endothelial layers of sinusoidal structure and finally hematopoietic repopulation.These findings pointed out that CFU-F include a group of cells with the capacity ofmultipotent differentiation into mesenchymal lineages. Based on these features, multipotentCFU-F were renamed 'mesenchymal stem cells' by A. I. Caplan in 1991 [48]. Caplan latercommented that the term 'stem cell' was provocative at that time but justified by ongoingresearch displaying that CFU-F could generate bone, cartilage, fat, muscle and othermesodermal phenotypes in vitro [49]. Postnatally, the contribution of BM-MSC to boneformation is associated with declining numbers of MSC after birth as indicated by the 10folddrop of CFU-F colony numbers in BM obtained from newborn and skeletally developedteenaged donors and a steady further decline with aging [50].Thus, bone formation was considered a core function of BM-MSC in vivo and theirosteogenic potential has been investigated in further detail by using scaffolds. As an example,porous calcium phosphate ceramic cubes of 3 mm in size were loaded with BM-MSCexpressing the genetic marker lacZ and then subcutaneously transplanted intoimmunodeficient mice [51]. The MSC monolayers formed osteoblasts, then the scaffoldbecame vascularized by host vessels and mineralized osteocytes developed. Importantly,lacZ+ osteoblasts and osteocytes confirmed that new bone was formed by donor MSC.Testings of this approach in animals and clinical settings showed that transplanted porousscaffolds loaded with BM-MSC significantly contributed to bone repair in rodents and inpatients with large bone defects [52, 53].The cube experiments further demonstrated that lacZ+ cells also occur around blood vessels[51]. In support, cultured CFU-F express the same markers [e.g. cluster of differentiation(CD) 146] as adventitial reticular cells of sinusoids in the intact BM in vivo [54].Consequently, BM-MSC which do not undergo osteogenic differentiation reside at theabluminal surface of endothelial cells. After subcutaneous transplantation, cells sorted forCD146 were capable to organize a hematopoietic microenvironment outside the BM. Thisconfirmed that skeletal progenitors are a functional part of the hematopoietic stem cell (HSC)niche and form a specialized microenvironment as conceptually already conceived by R.Schofield in 1978 [55]. The use of culture expanded BM-MSC to improve outcomes of BMACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT11transplantation in cancer patients after chemo-therapy was tested first-in-man in 1995 and wasclinically successful and safe [56].Lineage tracing, using nestin (Nes)- or leptin receptor (LepR) promoter driven expression ofthe fluorescence reporters GFP or tdTomato, was employed to further investigate MSCrelatedcell fates in the BM [57, 58]. One should keep in mind that characterization of BMlineages is complicated since marker expression like that of Nes-GFP is in part variableduring cellular development and overlapping between distinct phenotypes. Besides Nes andLepR gene activation, the in situ localization of MSC in the BM has been mainly defined bythe differential expression of CD146, CD271, neural/glial antigen 2 (NG2) and alpha smoothmuscle actin (α-SMA). Resulting findings indicated the existence of three different MSCpopulations within the endosteal niche and the perivascular niches at arterioles and sinusoids.These investigations disclosed that Nes+ MSC of the endosteal niche secrete factors orexpress cell surface molecules that regulate quiescence in nearby HSC [59, 60]. Nes+ MSC inthe perivascular niches express the key niche factors CXC chemokine ligand 12 (CXCL12),therefore also called CXCL12-abundant reticular (CAR) cells, and stem cell factor (SCF)which both control retention and maintenance of HSC [57]. The perivascular MSC could bedivided in rare periarteriolar NG2+ cells with high nestin expression (Nesbright/NG2+) andabundant perisinusoidal LepR+ cells with low nestin expression (Nesdim/Lepr+). Deletion ofCxcl12 and Scf in Nes+ MSC results in the mobilization of HSC to extramedullary organs anda marked reduction of HSCs in the BM [57, 61, 62]. Notably, the secretion of CXCL12 is inpart regulated by direct innervation of the sympathetic nervous system and modulated bycircadian rhythms [63].The very low numbers of CFU-F in BM of adult human donors points out that BM-MSC are aminor population [50]. In line with this, it was found that among BM-MSC 'abundant'CAR/LepR+ cells account for only 0.3% of mouse BM cells [64]. This small population is themajor source of adipocytes and osteoblasts in adult mouse BM but most of these cellsremained undifferentiated to maintain the hematopoietic niche. The underlying molecularregulation was unclear until recently Seike et al. found that CAR/LepR+ cells preferentiallyexpress early B-cell factor (Ebf) 3 and analyzed its function [65]. Deletion of Ebf3 inCAR/LepR+ cells severly impaired HSC niche function and BM became osteosclerotic withincreased bone in aged mice. Additional deletion of Ebf1 further increased niche dysfunctionACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT12leading to depletion of HSC already in infant marrow. This demonstrated that CAR/LepR+MSC-derived Ebf3 and Ebf1 are required to maintain the HSC niche by inhibition ofosteogenesis.Pericyte-MSC outside the BMThe early view on MSC as BM stroma cells has nowadays completely changed and theoccasionally used term 'mesenchymal stromal cell' became misleading. Instead, it becameobvious that MSC within the BM are not part of the connective tissue stroma but are formingthe endosteal and perivascular niches. Most BM-MSC are of perivascular origin [54]. In alandmark study, Crisan et al. clearly documented that MSC phenotypes exist outside the BMin multiple organs as perivascular pericytes expressing typical BM-MSC markers like CD146,NG2 and α-SMA, and being multipotent for osteogenic, chondrogenic, adipogenic andmyogenic lineages in vitro [66]. Further functional characterization tested whether suchpericyte-MSC possess the ability of BM-MSC to restore a hematopoietic niche in irradiatedmice [67]. The study revealed that sorted CD146+ perivascular cells, isolated from humanadipose tissue, are capable to support the long-term persistence of transplanted human HSCwhile CD146- perivascular cells did not. This observation clearly demonstrated that BM-MSCand pericytes expressing the same markers are equivalent in function.Various studies have meanwhile demonstrated that MSC phenotypes could be isolated fromvirtually all tissues of the body including fat, muscle, cord blood, Wharton's jelly, placentaand others [68]. This initiates the notion that possibly a unique MSC may exists but it becameobvious that all these MSC, beside core markers, display differential gene expression profilesin a time and tissue-related manner and thereby affect stemness [69]. For example, musclepericytes are not spontaneously osteochondrogenic while cord blood-derived MSCphenotypes display the unique capacity to form cartilage spontaneously in vivo. Furthermore,there is evidence that also the intrinsic mechanical properties of the extracellular matrixinfluences cell fate decisions in MSC, as softer matrices that mimic muscle are myogenicwhile rigid matrices that mimic collagenous bone are osteogenic [70]. Collectively, this raisesthe notion that mesenchymal stemness of MSC is adapted to or imprinted by tissuemicroenvironment and that MSC from placenta, Wharton's jelly, umbilical cord blood etc.may display the most embryonal-like phenotype [68, 69].ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT13Regarding the notion of developmental and tissue-related differences, Chen et al. recentlyproposed the concept of multiple 'paralogous' stem-cell niches which are progressively andfunctionally transformed within an individual organism throughout its life span [58]. In theirview, delineation of distinct cell phenotypes results from complex multiple interchangeableevents of epithelial to mesenchymal transition (EMT) and reverse mesenchymal to epithelialtransition (MET). These dynamic processes make it difficult to discern cell identities and todefine reliable markers. Therefore, the question whether all pericytes give rise to MSC, or inthe alternative view, pericyte-MSC differ from BM-MSC but may derive from a commonprogenitor, is not finally answered to date [49, 71, 72]. Surely, the answer will be complexand limited by the accuracy and composition of available marker sets; 'true' pericyte-MSCmay likely represent a subpopulation among all pericytes.In vivo function of pericyte-MSCPericytes have been discovered in the early 1870s by C.J. Ebert and C.M.B. Rouget [73] andwere named by K.W. Zimmermann describing their contractile nature in 1923 [74]. Latercharacterization specified that pericytes communicate with endothelial cells by both physicalcontact and secreted factors to regulate growth, stability, architecture and blood flow ofmicrovessels as well as they are important for the integrity of the blood brain barrier andprovide clearance and phagocytosis in the brain [75].Pericytes attach to the epithelium by their tips and their contractile apparatus consisting ofmicrofilaments containing actin, myosin and tropomyosin enables them to regulate thecapillary diameter or to move along the microvessels [76]. This indicated that perivascularpericyte-MSC and their BM counterparts are not static but dynamic and their close proximityto the vasculature enables them to readily mobilize and travel in the bloodstream to sites ofinjury. Consistent with this view, pericytes respond to a series of pro-inflammatory stimuliand are able to sense different types of tissue trauma signals by their expressed functionalpattern-recognition receptors and contribute to the onset of innate immune responses by cellcellcontact and paracrine effectors [73]. Similarly, transplanted BM-MSC home to varioussites of injury, e.g. stroke [77], pancreatic islet inflammation and diabetic kidney [78, 79] andcancer [80]. Once on site, BM-MSC secrete a variety of immunomodulatory, antiinflammatory,angiogenic, anti-apoptotic and tissue-regenerative trophic factors [81], andfend off invading microbes by secretion of anti-microbial peptide LL36 that kills bacteriaupon contact [82, 83].ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT14Altogether the ability of migration and humoral tissue restoration is a common feature ofMSC independent of their BM or pericyte origin. A.I. Caplan, who once coined the termMSC, has meanwhile suggested to rename these cells 'medicinal signalling cells' to moreaccurately reflect the new conceptional view on MSC as a 'drug store' for injured tissues invivo and to preserve the MSC nomenclature [84].MSC in cancerBesides MSC and HSC, the perivascular niche also accommodates tumour SC and itsmicroenvironment has been shown to regulate tumour dormancy and growth [85-87].Tumours recruit pericytes by e.g. platelet-derived growth factor (PDGF) to maintain theirtumour vessels [88] and consequently, inhibition of PDGF receptor signalling causes pericytedetachment and vessel regression, and diminishes tumour growth in several cancer models[89-91]. Tumour cells further interact with the surrounding stroma leading to a chronicallyincreased release of inflammatory cytokines and growth factors [92] that has been describedas a 'wound that never heals' [93]. The chronic inflammatory state drives the recruitment ofresponsive cell types including MSC [94, 95] which account for 0.01–1.1% of total cells inprostatectomies from human prostate tumours [96].It is now understood that MSC interact with tumour cells at various stages of progression butit is not finally clear whether their role is tumour promoting or suppressive. Several cancermodels implicated that MSC promote tumour progression and invasiveness as well as havinga role in the creation of a metastatic niche at the secondary site [97-100]. In contrast, MSCsuppressed tumour growth in several cancer models including breast cancer, Kaposi’ssarcoma, hepatoma and melanoma [101-104]. Reasons for conflicting findings may resultfrom the heterogeneity of tested MSC populations, differences in experimental design andvarying responses dependent on the stimuli [105].However, it seems unlikely that transplanted MSC have a significant role in inducing orpromoting tumours in human subjects, as their clinical use has been considered safe since1995 [56] and clinicians did not notice a tumour risk. In support of this notion, a metaanalysishas studied 1012 participants who received MSC for treatment of ischemic stroke,ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT15Crohn’s disease, cardiomyopathy, myocardial infarction, graft versus host disease or served ashealthy volunteers but did not find any indication of malignancy [106].Meanwhile, research has employed the recruitment of MSC to tumours in order to targetmalignant diseases with genetically modified MSC that, for example, overexpress pigmentepithelium-derived factor (PEDF) to reduce angiogenesis or overexpress TNF-relatedapoptosis inducing ligand (TRAIL) to induce apoptosis [107]. Despite using MSC fromdifferent sources, different transfection methods and a wide array of expressed proteins, thedata consistently showed a reduction in tumor growth and prolonged survival in rodents.These promising pre-clinical outcomes initiated the first-in-man trial TREAT-ME-1 whichaimed to target advanced gastrointestinal cancer (ClinicalTrials.gov: NCT02008539).The trial used ganciclovir (GCV) in combination with autologous BM-MSC overexpressingherpes simplex virus thymidine kinase (HSV-Tk) under the control of the CC-chemokineligand 5 (CCL5/RANTES) promoter. Mechanistically, engineered MSC migrate to tumourswhere they become activated to express CCL5 [98]. Subsequently induced HSV-Tkphosphorylates GCV which then inhibits DNA polymerases and thereby induces apoptosis intransfected cells and, due to a bystander effect, also in nearby tumour and stromal cells [108].The primary study aim was to evaluate safety and tolerability, and both features were foundgenerally favorable with stable disease in four patients, and progressive disease in 2 patientsafter one year follow-up [109]. Slowed tumour progression and enhanced survival are of greatimportance in the field and engineered MSC may contribute in the future to prolong the life ofcancer patients. Side note: the HSV-Tk/GCV suicide gene technique has also been tested asan 'emergency switch' that would allow to eliminate transplanted iPSC in case of malfunction[110].Transdifferentiation of MSC into insulin producing pancreatic β-cellsThere was initially great optimism that MSC could be easily transdifferentiated across thegerm layer border into insulin producing pancreatic β-cells and thereby avoid the ethical andtumorigenic obstacles of ESC and iPSC. Generation of insulin producing cells from MSCemployed genetic engineering including overexpression of PDX1, neurogenin 3 (NGN3) andpaired box 4 (PAX4) [111-113] and/or complex in vitro protocols using various conditionsand factors to resemble pancreatic development [114-118]. Depletion of β-cells in rodents bya high dosage of the β-cell toxic compound streptozotocin (STZ) has frequently been used toACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT16test the functional capacity of transplanted insulin producing cells. As a variant related toaspects of T2DM [119], multiple low-dose STZ-treatment causes islet inflammation fortesting of MSC-mediated recovery of β-cell dysfunction and partial loss. Aspects related tothe immunology of T1DM have been investigated in female NOD mice with spontaneouslyoccurring autoimmune insulitis [120].Several studies reported that transplanted MSC-derived insulin producing cells can improveglycemia in STZ-diabetic rodents [111, 114, 118, 121]. Nevertheless, stem cell specialistsremained sceptic concerning in vitro transdifferentiation of MSC beyond mesodermallineages. In this regard, the efficiency of MSC transdifferentiation was generally very low andresulting insulin producing phenotypes frequently possessed an acurate secretory capacity butwere not further expandable or vice versa. To date, transdifferentiation of MSC has notreached clinically significant large scale production of pancreatic progenitors or β-cells as ithas been established for ESC and iPSC.The study of Ianus et al. initiated the notion that injected BM cells contain a subpopulation ofcells that engraft into islets and are capable to transdifferentiate into insulin producingphenotypes in vivo [122]. It was observed that injection of BM cells with insulin gene 2promoter driven GFP expression into sublethally irradiated mice gave rise to a smallproportion of 1.7-3% glucose-responsive GFP+/insulin+ cells within islets which, afterisolation and sorting, show a functional insulin secretion comparable to control β-cells.Insulin+ phenotypes could be reproduced in a subsequent study. Hess et al. transplanted BMcells from GFP mice in NOD/SCID mice with multiple low-dose STZ-induced isletinflammation and noted partial recovery of diabetic blood glucose levels [78]. GFP-BM-cellssignificantly migrate to the inflamed endocrine pancreas and their occurrence within isletswas associated with enhanced local proliferation and 2.5% GFP+/insulin+ cells. Since theinsulin+/GFP+ cells did not express PDX1, a major marker of a mature and functional β-cell,the authors concluded that amelioration of hyperglycemia was not caused by incompletelydifferentiated GFP+/insulin+ cells but by the proliferative increase in β-cell mass.In further testing, using injection of GFP-BM cells into single-dose STZ-treated mice, only 2GFP+/insulin+ cells out of more than 100,000 screened β-cells could be retrieved [123].These very rare events were considered to rather result from cell fusion thantransdifferentiation [124, 125]. Importantly, an elaborated lineage tracing study from DouglasACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT17Melton’s group strongly suggested that new β-cells and islets only derive from pre-existing β-cells and not from adult pancreatic stem cells or progenitors [126]. In this regard, a later studyreported that up to 3% of injected human BM-MSC engrafted into inflamed pancreatic isletsof multiple low-dose STZ-diabetic NOD/SCID and improved hyperglycemia by reduction ofβ-cell loss and partly maintained mouse insulin blood levels in the absence of detectablehuman insulin [79]. In addition, up to 11% of injected human BM-MSC engrafted into theSTZ-injured kidneys and improved glomerular morphology as well as decreased mesangialthickening and macrophage infiltration.Collectively, these studies exclude significant transdifferentiation of MSC into insulinproducing cells in vivo and pave the way for the new understanding that MSC migrate to andengraft at site of injury to support tissue repair by secretion of numerous tissue regenerativefactors [81].Humoral potential of MSCAfter isolation, pancreatic islets suffer from hypoxic culture stress due to loss of blood supplyand consequently impeded transport of oxygen to the inner cell layers of the threedimensionalislet structure [127, 128]. After transplantation, islets were further challenged bylocal inflammation and rejection processes [129]. Fast dynamics of revascularisation anddownregulation of immune responses have been considered important for long-term graftfunction and generated interest on the angiogenic and immunomodulatory potential of MSCin the context of islet transplantation.Kinnaird et al. displayed that human MSC express a wide array of arteriogenic cytokine genesand that MSC conditioned media promoted smooth muscle cell proliferation and migration ina dose-dependent manner in vitro [130]. In vivo, using a murine hindlimb ischemia model,murine MSC conditioned media enhanced collateral flow recovery and remodeling, improvedlimb function, reduced the incidence of autoamputation, and attenuated muscle atrophycompared with control media. In this regard, Figliuzzi et al. tested the angiogenic effects ofBM-MSC on co-transplanted islets in STZ-diabetic rats and noted that improved graftsurvival and function in association with increased numbers of new capillaries and expressionof vascular endothelial growth factor (VEGF) [131]. Upcoming studies confirmed theangiogenic capacity of MSC and its association with VEGF [132-135]. In this regard, it wasshown in vitro that VEGF inhibition partially blocked the enhanced formation ofACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT18anastomosing tubule networks by co-cultured endothelial cells [134]. Therefore, VEGFappears to be an important player which is supported by other MSC-derived factors such asnerve growth factor (NGF) [136] and factors inducing angiopoietin receptor Tie-2 expressionin islets [135]. In sum, these studies established that MSC-mediated revascularisationcontributes to islet graft survival by shortening the post-transplantation ischemia period.Improved revascularization and functional outcome of co-transplanted islet grafts have beenfurther associated with reduced numbers of terminal deoxynucleotidyl transferase mediateddUTP nick end labeling (TUNEL)+ and caspase-3+ apoptotic cells [137, 138]. AngiogenicVEGF and several other MSC-released trophic factors including hepatocyte growth factor(HGF), insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β, basicfibroblast growth factor (bFGF) and granulocyte-macrophage colony-stimulating factor (GMCSF)display anti-apoptotic properties [81, 139]. The potential of MSC to mediate survivalwas tested by direct interactions with β-cells in vitro in the absence of third party cells fromsurrounding tissues.In line with the variety of released growth factors, we and others displayed that MSCconditionedmedium or co-cultured MSC preserve Akt signaling in cultured islets undergoinghypoxic culture stress and additional treatment with alloxan and STZ [135, 140]. Aktsignaling promotes survival and reduces intrinsic apoptosis by its influence on B-celllymphoma 2 (Bcl-2) family proteins such as phosphorylation of Bcl-2 antagonist of cell death(BAD) and caspase-9 [141]. MSC-released factors also activate mitogenic extracellularsignal–regulated kinases (ERK)1/2 signaling which, similar to Akt, promotes survival byinhibition of intrinsic apoptosis [142, 143]. Interestingly, MSC induced ERK1/2 signalingonly in highly proliferative endothelial cells and INS-1E insulinoma cells but not in primarymouse islets with a low proliferation rate [135, 140, 144]. These observations indicate animportant role for the Akt pathway in MSC-mediated survival of pancreatic islets.MSC-released factors have been further reported to improve insulin secretion and glucoseresponse(see Table 1 in [145]). Experiments may indicate a beneficial effect of cell-cellcontacts since humoral improvement of glucose-stimulated insulin secretion (GSIS) inindirect co-cultures with cells separated by membranes [135] was not well reproducible byother studies unless cells were cultured in direct contact [146, 147]. In this respect, MSCenhance GSIS in vitro by release of annexin-1 (ANXA1) while MSC from Anxa1-/- mice hadACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT19no functional capacity [148]. Hence, heterogeneous effects of MSC on GSIS may partly resultfrom different expression levels of ANXA1. Likely, the very close proximity of MSC andislets in direct co-cultures enhanced local effector levels and involves the extracellular matrix(ECM) since MSC co-cultured with HSC maintain the vascular niche by upregulatedexpression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule1 (VCAM1) [149]. Such supportive processes are an important topic in tissue engineering andit has been recognized that islets, which lost ECM during enzymatic isolation, show improvedsurvival and function after treatment with ECM molecules [150].Moreover, numerous MSC-released factors exhibit potent immunomodulatory characteristics;e.g. transforming growth factor-β1 (TGF-β1), indoleamine 2,3-dioxygnase (IDO), nitric oxide(NO), human leukocyte antigen-G (HLA-G), Prostaglandin E2 (PGE2), interleukin-1 receptorantagonist (IL-1RA) and tumour necrosis factor-stimulated gene 6 (TSG-6) [81, 139, 151]. Asa result, MSC have been described to induce regulatory T cells and anti-inflammatory M2macrophages, and to inhibit T cells, natural killer cells and T helper (Th)17 cell differentiationas well as maturation of dendritic cells (DC).Consequently, MSC substantially reduced co-transplanted islet graft inflammation andrejection in BALB/c mice [138, 152], humanized NOD scid gamma (NSG) mice [153] and acynomolgus monkey model [154]. All these studies showed MSC-improved engraftment inassociation with reduced infiltration of T cells and neutrophils, and increased numbers ofcirculating regulatory T cells. Inhibitors established that MSC-mediated prevention of T cellproliferation and islet graft rejection was not related to IDO and heme oxignase-1, partiallyrelated to NO and profoundly mediated by matrix metalloproteases (MMP)-2 and MMP-9 viareduction of IL-2 receptors on T cells [152]. MSC further suppress the proliferation andactivation of T cells by interaction with IL-10-producing CD14+ monocytes [153].Remarkably, systemically injected MSC in female NOD mice reduced the incidence ofspontaneous T1DM [155] or reversed recent-onset hyperglycemia via release of programmedcell death ligand 1 (PD-L1) and inhibition of myeloid/inflammatory DC through an IL-6-dependent mechanism [156]. Moreover, treatment of NOD mice with CD4+CD62L+regulatory T cells (Treg), which have been cocultured with cord blood-derived MSC before,resulted in a marked reduction of spontaneous autoimmune insulitis, restored Th1/Th2cytokine balance in blood and induced apoptosis of infiltrated leukocytes in pancreatic isletsACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT20[157]. This concept has been translated into clinic as 'Stem Cell Educator' therapy (see below)[158, 159].In addition, MSC have been further tested for their ability to ameliorate wound healing whichis a frequent diabetic complication. Endogenous MSC, present in the skin as dermal sheathcells surrounding hair follicle units [160] and as perivascular pericytes [161]. Skin injuryinduces MSC to recruit and activate epithelial cells, fibroblasts and keratinocytes torevascularize and re-populate the wounded area during the proliferative healing phase [162].Wounds treated with MSC show acceleration of angiogenesis and re-epithelialisation [163]which, according to the notion of paracrine factors, could also be achieved by treatment withMSC-conditioned medium [164-166]. Wound healing is impaired in DM patients which showdegraded micro- and macrovessels in association with early occurring detachment and loss ofvascular pericytes at capillaries [167, 168]. Importantly, MSC-treatment successfullyimproved wound healing under diabetic conditions such as in diabetic db/db mice withmutated leptin receptor [169] and a rat model of diabetic foot ulceration [170].MSC in clinical trialsThe complex and wide-ranged humoral potential of MSC attracted much attention amongresearchers and clinicians. MSC can be isolated from various tissues, frequently from BM andadipose tissue, by minimal invasive puncture and they also allow noninvasive retrieval fromoften discarded 'medical waste' such as placenta, cord blood and umbilical cord [68, 171]. It isfurther known from early CFU-F studies and numerous studies since then that MSC could beeasily expanded in vitro without significant loss of their mesenchymal differentiation capacityor their humoral secretion. Moreover, MSC are immune-privileged because they express verylow levels of major histocompatibility complex (MHC) class I and no MHC class II whichnormally prevents or strongly reduces immune responses [172, 173]. In clinical use since1995 [56], MSC are considered clinically safe [106] and both administration of autologousand also allogenic MHC-mismatched MSC is generally well tolerated and clinically effective[174-176].To date, CinicalTrials.gov listed over 850 therapeutic approaches using MSC to target a broadarray of diseases including hematological disease, graft-versus-host disease (GVHD), organtransplantation, cardiovascular and neurological diseases, bone and cartilage repair as well asinflammatory and autoimmune diseases [177]. Among these, more than 60 trials addressACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT21T1DM and T2DM, and from these we have summarized all trials with reported outcome inTable 1. In these trials, the various humoral features of MSC address different disease aspects(Fig. 1). In many patients with T1DM a minor portion of insulin producing β-cells survive butcan not recover unless thereby induced autoimmune responses are blocked [178]. MSCmediate immune tolerance that aims to enable partial recovery of remaining β-cell mass [158,159] or to reduce and delay the β-cell destruction during new-onset of T1DM [179, 180]. InT2DM, the anti-inflammatory features of MSC were used to ameliorate chronic low-gradeinflammation which has been recognized as an important cause of insulin resistance and β-cell dysfunction [119]. These features in combination with secretion of pro-angiogenic factorsshould improve engraftment and survival of transplanted islets [181].Interestingly, there is only one completed trial investigating the effect of MSC cotransplantationon islet graft survival and function. Potentially, there are concerns on theadditional expense needed for generation, testing and application of clinical-grade MSC sinceestablished immunosuppression regimes should prevent graft rejection. In this regard, Wanget al. tested combined autotransplantation of BM-MSC and islets in chronic pancreatitispatients undergoing pancreatectomy without immunosuppression [181]. Patients showedreduced insulin requirement in the peritransplantation period, reduced decline of C-peptidelevels after 6 month and lowered fasting blood glucose levels after 12 month. This suggeststhat co-transplanted MSC reduced loss of islet graft function. Additional studies and longtermobservations are needed to verify these very limited results from 3 patients.The other studies addressed T1DM (7 trials) as well as severe T2DM (8 trials) in patients whorequired insulin and/or oral anti-diabetic drugs to control glycemia. Currently the total numberof investigated patients is relatively small. In total, 276 patients were investigated in smallgroups of 6-22 subjects and 3 studies [159, 174, 182] analyzed groups of 31-45 subjects. Theenrolled patients show diversity regarding age, BMI and other aspects as well as duration andsevereness of the disease. In this view, also applied MSC came from different sourcesincluding BM, adipose tissue and umbilical cord, have been differentially processed andapplied in different dosages. Though MSC did not cure the disease and despite muchheterogeneity regarding applied MSC, it is quite astonishing that studies reported varyingpositive aspects of partially improved glycemia. Only two T2DM trials reported on diabeticcomplications. Jiang et al. noted without further details that renal and cardiac functionsshowed varying degrees of improvement [183] and Hu et al. found no rise of diabeticACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT22complications in MSC-treated patients while placebo-treated patients displayed higherincidences of diabetic retinopathy, neuropathy and nephropathy during 36 months follow-up[182]. In general, MSC were well tolerated and it could be noted as the quintessence ofoutcome that all trials except one [179] reported reduced requirement for exogenous insulinand/or anti-diabetic drugs.Two studies addressed the question whether MSC treatment could delay development ofnewly-onset T1DM. Hu et al. reported that both the HbA1c and C-peptide were improvedcompared to the pre-therapy values and to control patients during 2 years follow-up [180].Consequently, MSC-patients required smaller insulin dosages. This outcome indicated areduced loss of insulin producing β-cells but was not fully reproduced by Carlsson et al. whoobserved improved C-peptide levels in response to a mixed-meal tolerance test but no changesin HbA1c, fasting C-peptide and daily insulin dosages after 1 year [179]. More trials withprolonged observation periods are needed to clarify the potential of MSC to delay thedevelopment of T1DM.An interesting aspect is the so-called 'Stem Cell Educator' [158, 159]. While studies normallyapply MSC by intravenous injection, the 'Stem Cell Educator' approach routed the patient'sblood through a closed-loop system that separates lymphocytes from the whole blood andbriefly co-cultures them with adherent cord blood-derived MSC before returning them intothe patient’s circulation. Though MSC are not delivered into the body, their temporary contactto patient's lymphocytes was sufficient to induce immune tolerance which ameliorates thedisturbed Th1/Th2/Th3 cytokine balance with increased Treg numbers in type 1 diabeticpatients and decreased CD86+/CD14+ monocytes and reduced markers of inflammation intype 2 diabetic patients. As a result, all patients displayed a generally reduced requirement forinsulin and metformin and improved HbA1c values after 10-12 months follow-up. In addition,homeostasis model assessment of insulin resistance (HOMA-IR) demonstrated that insulinsensitivity was improved post-treatment in type 2 diabetics. The potential of the 'Stem CellEducator' is currently further investigated by 2 further trials (ClinicalTrials.com:NCT02624804 and NCT03390231).Collectively, the trials show that even heterogeneous MSC populations could be clinicallyeffective. The question, however, remains whether clinical outcome could be improved byoptimized MSC batches since actually no standardized method exists for isolation,ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT23characterization, expansion, potency testing or pathogen screening [184-186]. A basic issue isthe donor heterogeneity which has the potential to dramatically influence therapeuticalproperties of MSC. This includes the question whether autologous MSC are a good choice forcell therapy of DM since, for eample, hyperglycemia induces pericyte dysfunction viaactivation of p75 neurotrophin receptor/NF-kB-mediated release of microparticles carryingmiR-503 from neighbouring endothelial cells [187]. Also subclinical inflammation present insubjects with metabolic syndrome and T2DM [119] impairs the vascular stem cell niche andleads to MSC dysfunction [188]. MSC batches could be further influenced by isolation andexpansion since several studies suggested that outgrowth from donor tissue generates lessheterogeneous cell populations with increased proliferation rates and cell viability thanisolation by enzymatic tissue digestion [189-191]. Moreover, MSC may lose their functionsdue to increased cellular senescence during longer expansion and passaging since a directpositive link between early passages of MSC and clinical outcomes in GVHD has beendemonstrated [192]. One should finally keep in mind that only a small proportion ofsystemically injected MSC engraft at site of injury while most rapidly embolise in the lungsand disappear with a half-life of about 24 h to an unclear fate [193, 194].The International Society for Cell Therapy (ISCT) defined clinically useful MSC bymesenchymal differentiation (into bone, cartilage and fat), plastic-adherent growth in vitroand expression of CD73, CD90 and CD105 in the absence of hematopoietic surface markers[195]. Notably, expansion of plastic-adherent BM cells favours the expansion of nonclonalstromal cell-enriched populations, often misinterpreted as pure SC fractions, which containvarying percentages of true MSC, e.g. depending on donor age [50], and thereby plausiblyexhibit different clinical effectiveness [196, 197]. This demands a reliable assay, such as theCFU-F assay, and careful evaluation of each MSC batch may allow the identification of thepercentage of stem cells and their multilineage potential in each batch of nonclonal MSC.Potentially, efficacy of MSC populations could be further enhanced by selection viaadditional markers such as stromal (STRO)-1, CD146, alkaline phosphatase, CD49a, CD271and VCAM1 [197]. Against this background, the US Food and Drug Administration (FDA)demands registering of tissue processing facilities which should report on (i) prevention oftransmitting communicable disease via contaminated tissue, (ii) proper handling andprocessing of tissue and (iii) demonstration of clinical safety and effectiveness of cells,especially after extensive manipulation.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT24Conclusion on the current state of stem cell therapy of diabets mellitusStem cell therapy has to deal with a wide array of limitations which are still the subject ofcurrent research. See Table 2 for summary and comparison of SC in cell therapy of DM.Differentiation of ESC and iPSC has meanwhile reached clinical large-scale production andcurrent developments of macroencapsulation may provide clinical safe usage of these cellsthat demonstrate otherwise potentials for tumor development. Macroencapsulation preventsthe escape of embedded cells into the body and subcutaneously transplanted devices could beretrieved and removed easily. The outcome of Viacyte's first-in-man trial of their pancreaticendoderm implant VC-01 will clarify whether the use of ESC and iPSC is an option in DMtherapy in the near future.MSC are clinically safe and several trials exist though they are limited in number andinvestigated patients. Currently, MSC-based therapy is no cure but shows a potential toameliorate DM since most studies report decreased requirement of exogenous insulin and/oranti-diabetic drugs. In this regard, MSC may be best used with diabetic patients that havesevere problems in controlling glycemia by conventional therapies; e.g. patients with brittleDM. We see potential in optimizing their therapeutical performance by standardizingisolation, characterization, selection, expansion, potency testing and pathogen screening.However, increased efforts will increase costs and MSC have to prove their value.Declarations of interestNone.Author contributionsAll authors have participated in manuscript preparation and have approved the final article.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT25References[1] Perakakis N, Mantzoros CS. Immune therapy in type 1 diabetes mellitus - Attempts tountie the Gordian knot? Metabolism: clinical and experimental. 2016;65:1278-85.[2] Pepper AR, Bruni A, Shapiro AMJ. Clinical islet transplantation: is the future finally now?Current opinion in organ transplantation. 2018;23:428-39.[3] Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islettransplantation in seven patients with type 1 diabetes mellitus using a glucocorticoidfreeimmunosuppressive regimen. 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Clinical trials with reported outcome using MSC to treat DM.Section A. T1DMReference,Trial number,Country,Patient number,DurationDiseaseObjectivePatient characteristics Treatment Main outcomesWang et al. 2018NCT02384018United Statesn = 312 monthsTesting safety and effects ofBM-MSC and isletautotransplantation inpatients with chronicpancreatitis undergoing totalpancreatectomyAged 26, 29 and 42years with chronicpancreatitisInfusion of 20 ± 2.6 x 106BM-MSC together with5,107 ± 1,920 isletequivalents/kg via theportal vein101 patients withpancreatectomy due tochronic pancreatitis whoreceived islets aloneserved as historicalcontrolsNo AE directly related to MSCInsulin requirement lowered in the peritransplantationperiodFasting blood glucose loweredFasting C-peptide with smaller declinesduring months 1-6 (mean C-peptide levelscomparable to control values at 6 months)HbA1c not differentImproved life qualityCai et al. 2016NCT01374854Chinan = 2112 monthsTesting safety and effects ofcombined UC-MSC plusautologous BM-MNC withoutimmunotherapy on C-peptideAged 18-40 years withestablished T1DM for2-16 yearsHbA1c 7.5-10.5%Fasting serum C-peptide< 0.1 pmol/mlDaily insulin requirement< 100 IUInfusion of UC-MSC (1.1 x106/kg) and BM-MNC(106.8 x 106/kg) throughsupraselectivepancreatic arterycannulationControls receivedstandard care (n = 21)MSC-treatment was well tolerated1 patient with puncture site bleeding1 patient with abdominal painSerum C-Peptide AUC 105.7% increase,controls 7.7% decreaseSerum insulin AUC 49.3% increase, controls5.7% decreaseHbA1c decreased 12.6%, controls increasedby 1.2%Reduced insulin requirement (-29.2%) withno change in controlsCarlsson et al. 2015 Testing safety and effects of Aged 18-40 years IV infusion of 2.1–3.6 x No side effects of MSC treatmentACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT41NCT01068951Swedenn = 912 monthsautologous BM-MSC intreatment of patients withrecently diagnosed T1DMT1DM newly diagnosed< 3 weeks beforeenrollmentMMTT-stimulated serumC-peptide ˃ 0.1 nmol/l106 autologous BMMSC/kgSham controls (n = 9)HbA1c, fasting C-peptide and insulinrequirements not different compared tocontrolsMMTT-induced C-peptide AUC and peakvalues were preserved/increased by MSCDave et al. 2015Indian = 1036 monthsTesting co-infusion of ISC (invitro differentiated fromadipose tissue-derived MSC)together with BM-HSCAged 8-45 yearsIDDM for at least 6monthsC-peptide levels < 0.5ng/mlInfusion into portalcirculation, thymus andsubcutaneous tissueISC: mean of 3.34 ml cellinoculums with 5.25 x104 cells/μl; ISCexpressed ISL1, PAX6and IPF1 with mean Cpeptideand insulinsecretionof 1.03 ng/mland 17.48 l IU/l after 2 hin glucose mediumHSC: mean of 103.5 mLwith 2.66 x 106/μLNo untoward effect of stem cell infusionImproved serum C-peptide, Hb1Ac, bloodsugar levelsReduced exogenous insulin requirementPatients returned to normal lifestyle andunrestricted dietThakkar et al. 2015Indian = 2024 monthsTesting co-infusion of ISC (invitro differentiated fromadipose tissue-derived MSC)together with BM HSCComparison of autologous vs.allogenic stem cellsAged 8-45 yearsGroup 1: mean age 20.2years, mean diseaseduration 8.1 yearsGroup 2: mean age 19.7years, mean diseaseduration 7.9 yearsInfusion into portalcirculation, thymus andsubcutaneous tissueGroup 1 received 2.65 ±0.8 x 104 autologousISC/kg (n = 10); ISCexpressed ISL1, PAX6and IPF1Group 2 received 2.07 ±0.67 x 104 allogenicISC/kg (n = 10)HSC not reportedNo untoward effect, morbidity (pulmonaryembolism, sepsis) or mortality caused bytherapyReduced insulin requirementSustained improvement in serum C-peptideand HbA1cHu et al. 2013ChinaTesting the long-term effects ofWJ-MSC for newly-onsetT1DMAged 17.6 ± 8.7 yearsT1DM according to ADAcriteria for less than 62 IV infusions with a meanof 1.5-3.2 × 107 WJMSCwith an interval ofNo acute or chronic side effects compared tocontrol groupFasting plasma glucose levels not differentACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT42n = 1524 monthsmonthsFasting C-peptide ≥ 0.3ng/ml1 monthControl group receivedsaline (n = 14)from controlsImproved HbA1c, fasting C-peptide andpostprandial blood glucose levelsReduced insulin requirementZhao et al. 2012NCT01350219China, Spainn = 1210 monthsTesting safety and efficacy ofStem Cell Educator therapyAged 15 to 41 yearswith a diabetic historyof 1 to 21 yearsGroup A: with someresidual pancreatic β-cell function (n = 6)Group B: without (n = 6)Stem Cell Educatortherapy: patient’s bloodcirculated through aclosed-loop system thatseparates lymphocytesfrom the whole bloodand briefly co-culturesthem with adherent CBMSCbefore returningthem to the patient’scirculationNo AE, minimal pain from two venipuncturesReduced insulin requirement (24 weeks)Improved fasting C-peptide levels andreduced HbA1C (12 weeks) in groups Aand BIncreased in basal and glucose-stimulated Cpeptidelevels (40 weeks, group B)Increased expression of co-stimulatingmolecules CD28 and ICOS, increasednumbers of Tregs and restoredTh1/Th2/Th3 cytokine balance (4 weeks,groups A and B)Sham controls (n = 3) without significantchangesVanikar et al. 2010Indian = 1112 monthsTesting co-infusion of allogenicISC (in vitro differentiatedfrom adipose tissue-derivedMSC) together with BM HSCAged 5-45 yearsIDDM for 1-24-yearsLow serum C-peptidelevels < 0.5 ng/mlIntraportal infusionISC: mean of 1.5 ml with2.1 x 103/μL; ISCexpressed ISL1, PAX6and IPF1HSC: mean of 96.3 mlwith 28.1 × 103/μLNo adverse side effect related to stem cellinfusion or therapyReduced insulin requirementHbA1c decreasedSerum C-peptide levels increasedPatients became free of diabetic ketoacidosiseventsSection B. T2DMReference,Trial number,Country,Patient number,DurationDiseaseObjectivePatient characteristics Treatment Main outcomesBhansali et al. 2017 Comparison of safety and Aged 30-60 years with Infusion of 106 BM-MSC 1 patient with local extravasation of bloodACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT43NCT01759823Indian = 1012 monthseffects of autologous BMMSCand BM-MNC inreducing on insulinrequirementT2DM ≥ 5 years andfailure to achieve HbA1c≤ 7.5% while receivingtriple oral anti-diabeticdrugs in optimal dosesalong with insulin for thelast 6 months(group I, n = 10) or 109BM-MNC (group II, n =10) via transfemoralroute into the celiactrunkSham controls wereinfused into the femoralartery (group III, n = 10)following infusionReduced insulin requirement in BM-MSC andBM-MNC groups (6 of 10 patients in bothBM-MSC and BM-MNC groups, but none inthe control group achieved the primary endpoint of ≥ 50% reduced insulin requirement)Increased 2nd-phase C-peptide response inBM-MNC groupImprovement in insulin sensitivity index withincreased insulin receptor substrate-1 geneexpression in ABM-MSC groupHu et al. 2016Chinan = 3136 monthsTesting safety and long-termeffects of WJ-MSC onT2DMAged 18-60 years withT2DM according to ADAcriteria2 infusions of 106 WJ-MSwith an interval of 1month (group I) throughveins in the back of thehandControls (group II)received normal saline(n = 30)No serious AENo chronic side effects or lingering effectsFasting plasma glucose almost unchangedReduced insulin requirementFasting C-peptide improvedHbA1c and postprandial plasma glucoseimproved by trend after 36 monthsHOMA of β-cell secretory function improvedHOMA-IR unchangedIncidence of diabetic retinopathy, neuropathyand nephropathy only increased in controlsSkyler et al. 2015NCT01576328United Statesn = 453 monthsTesting safety, tolerability,and feasibility of allogeneicBM-derived STRO-3-selected subset of BMderivedMPC(Rexlemestrocel-L,Mesoblast Inc.; productexpressed MSC markersSTRO-1 and CD146) inT2DM inadequatelycontrolled with metformin orone additional oral anti-Aged < 80 years withT2DM for 10.1 ± 6 yearsHbA1c 7.0-10.5%Metformin either alone orin combination with oneother oral antidiabeticmedication (except athiazolidinedione) for atleast 3 monthsIV infusion of 0.3, 1 or 2 x106 BM-derived MPC (n= 15 each)Sham controls (n = 15)No serious acute AE due to infusionNo serious hypoglycemia events ordiscontinuations due to AE, comparable AEin MSC and placebo groups, 1 subject withsevere abdominal pain in MSC groupNo immunologic responses to MSCHbA1c reduced by trend, more pronouncedin patients with baseline HbA1c ≥ 8%Insulin requirement reduced by trendACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT44diabetic drugGuan et al. 2015Chinan = 624-43 monthsTesting safety and effects ofallogenic UC-MSCAged 27-51 yearsTime from hyperglycemiato first infusion was 4-157 weeksPatients treated withinsulin and poorlycontrolled blood glucoselevels and HbA1c2 IV infusions of 106 UCMSC/kg through thecubital vein with aninterval of 14-17 daysNo safety issues during infusion and thelong-term monitoring periodReduced insulin requirement (significantduring months 1-6), 3 patients becameinsulin-free for 25 to 43 monthsInsulin-free patients displayed reducedHbA1c and increased fasting C-peptideduring months 1-24Relative stable fasting plasma glucose and 2h postprandial blood glucose levelsLiu et al. 2014Chinese Clinical TrialRegister ChiCTRONC-10000985Chinan = 2212 monthsTesting safety and effects oftreatment with allogenicWJ-MSCAged 18–70 yearsT2DM according to ADAcriteriaPoor glycemic control withrecent antidiabetictherapies, includingdrugs and/or insulininjection for at least 3monthsGAD antibody negativeFasting blood glucoselevel ≥ 7.0 mmol/LHbA1c ≥ 7%2 infusions of 106/kg WJMSC1st infusion via peripheralvein on day 52nd infusion directlydelivered to thepancreas via the splenicartery usingendovascular catheterson day 103 patients with fever after operative day1 patient with subcutaneous hematoma1 patient with nausea, vomiting andheadacheImproved HbA1c, fasting C-peptide levels,HOMA of β-cell secretory function,postprandial blood glucose levelsReduced insulin requirement and oralhypoglycemic drugsReduced serum levels of IL-1β and IL-6, andreduced numbers of CD3+ and CD4+ Tlymphocyte numbers at 6 monthsKong et al. 2014NCT01413035Chinan = 186 monthsTesting safety and effects ofallogenic UC-MSCAged 23-65 yearsT2DM according to WHOcriteriaPatients received insulinand oral anti-diabeticdrugs to control3 IV infusions of 1-3 x 106UC-MSC/kg with aninterval of 1 week4 patients with slight transient fever8 patients respond to treatment (efficacygroup); these show: reduced fasting andpostprandial blood glucose levels, and bytrend increased plasma C-peptide levelsand Treg numbersAll patients had a feeling of well-being andare more activeZhao et al. 2013NCT01415726United StatesTesting safety and efficacy ofStem Cell EducatortherapyAged 29-68 years withlong-standing T2DMGroup A: oral medicationsStem Cell Educatortherapy: patient’s bloodcirculated through aNo AE, mild discomfort during venipuncturesImproved metabolic control and reducedinflammationACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT45n = 3612 months(n = 18)Group B: oral medications

Itinerary

insulin injections (n =11)Group C: impaired β-cellfunction with oralmedications + insulininjections (n = 7)closed-loop system thatseparates lymphocytesfrom the whole bloodand briefly co-culturesthem with adherent CBMSCbefore returningthem to the patient’scirculationReduced HbA1C in groups A and BImproved insulin sensitivity (HOMA-IR, 4weeks)Recovery of fasting C-peptide levels in GroupC (56 weeks) and HOMA of β-cell secretoryfunction/C-peptide (12 weeks)Improved serum TGF-β, reducedCD86+CD14+ monocytes, no effect onTreg numbers and restored Th1/Th2/Th3cytokine balance (4 weeks)Jiang et al. 2011Chinan = 106 monthsTesting safety and effects ofplacenta-derived MSC inpatients with longerduration of diseaseAged 30-85 yearsDuration of DM ≥ 3 yearsInsulin requirement foroptimal glycemic controlof ≥ 0.7 IU/kg/day atleast for 1 year3 IV infusions of 1.22-1.51x 106/kg placentaderivedMSC with aninterval of 1 monthNo fever, chills, liver damage and other sideeffectsInsulin and C-peptide levels increasedHbA1c decreasedInsulin requirement decreased, 4 of 10patients achieved reduction of ˃ 50%Improved renal and cardiac function (nodetails)Abbreviations. ADA, American Diabetes Association; AE, adverse event; AUC, area under the curve; BM, bone marrow; BMI, bodymass index; CD, cluster of differentiation; DM, diabetes mellitus; GAD, glutamic acid decarboxylase; HbA1c, glycated hemoglobin A1c;HOMA, homeostasis model assessment; HSC, hematopoietic stem cells; IDDM, insulin-dependent diabetes mellitus; IL, interleukin;ICOS, inducible costimulator; ISL-1, insulin gene enhancer protein 1; IPF1, insulin promoter factor 1 (official name, PDX1, pancreaticand duodenal homeobox 1); IR, insulin resistance; ISC, insulin secreting cell; IU, international unit; IV, intravenous; MNC, mononuclearcells; MMTT, mixed-meal tolerance test; MPC, mesenchymal precursor cells; MSC, mesenchymal stem cells; PAX6, paired boxprotein 6; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; TGF, transforming growth factor; Th, T helper cell; Treg,regulatory T cell; UC, umbilical cord; WHO, world health organisation; WJ, Wharton's Jelly.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT46Table 2. Summary and comparison of SC in cell therapy of DM.ESC iPSC MSCCell type and origin Embryonic SCInner cell mass of the blastocystAdult somatic cellsReprogramming in vitroAdult SCEndosteal (BM) and perivascular niches (alltissues)Characteristics PluripotentGenerates all germ layers:ectoderm, endoderm andmesodermPluripotentGenerates all germ layers:ectoderm, endoderm andmesodermMultipotentGenerates mesenchymal lineages: bone,cartilage, fat and muscleMaintain HSC niche and hematopoiesisEthical concerns Use of embryos No NoDifferentiation intopancreatic β cellsYesYesInsulin+ cells with limited secretory orproliferative capacity (experimental)Cell therapeutic options β-cell replacement β-cell replacementPatient-specific cell linesSecreted factors with immunomodulatory,angiogenic and tissue regenerative propertiesAdvantages Large-scale production ofpancreatic endoderm, endocrineprogenitors and fully functional β-cellsLarge-scale production ofpancreatic endoderm, endocrineprogenitors and fully functional β-cellsEasy isolation and in vitro expansionLow immunogenicity allows allogenictransplantation without immunosuppressionMinimal-invasive applicationClinical safe and well toleratedLimitations Tumorigenic if incompletelydifferentiatedTumorigenic if incompletelydifferentiatedSomatic mutationsIncomplete reprogrammingmaintains somatic transcriptionalmemoryISCT minimal criteria for clinical MSC favorexpansion of nonclonal stromal cell-enrichedpopulations with varying proportions of true SCCurrent clinical protocols are not standardizedand exhibit potential for improvementsOnly a small proportion of systemically injectedcells engrafts in injured target tissuesStatus First-in-man trial currentlyinvestigates clinical safety andefficacy of macroencapsulatedESC-derived pancreaticendodermMacroencapsulation avoidsCurrently not safe enough forclinical usagePatient-specific cell lines allowinvestigation of diseaseprocesses in vitro and represent aplatform for drug testingCompleted clinical trials collectively report onreduced requirement for exogenous insulinGreatest benefit for patients with problems incontrolling glycemia by conventional therapyMore clinical trials in progressACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT47tumorigenicity by preventing theescape of embedded cells into thebody and allows easy graftremoval if necessaryAbbreviations. BM, bone marrow; DM, diabetes mellitus; HSC, hematopoietic stem cell; ISCT, International Society for Cell Therapy;SC, stem cells.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT48HighlightsLarge-scale production of pancreatic endoderm and β-cells from embryonic stem cellsFirst-in-man trial investigates macroencapsulated embryonic stem cell-derived β-cellsInduced pluripotent stem cells allow patient/disease-specific cell linesMesenchymal stem cells secrete immunomodulatory and tissue regenerative factorsTransplanted mesenchymal stem cells ameliorate human type 1 and type 2 diabetes