Stem cells in the treatment of diabetes mellitus – focus on
mesenchymal stem cells

Stem cells in the treatment of diabetes mellitus

Stem cells in the treatment of diabetes mellitus – focus on
mesenchymal stem cells
– Günter Päth, Nikolaos Perakakis, Christos S. Mantzoros, Jochen
Seufert
PII: S0026-0495(18)30215-4
DOI: doi:10.1016/j.metabol.2018.10.005
Reference: YMETA 53824
To appear in: Metabolism
Received date: 10 August 2018
Accepted date: 14 October 2018
Please cite this article as: Günter Päth, Nikolaos Perakakis, Christos S. Mantzoros, Jochen
Seufert , Stem cells in the treatment of diabetes mellitus – focus on mesenchymal stem
cells. Ymeta (2018), doi:10.1016/j.metabol.2018.10.005
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Stem cells in the treatment of diabetes mellitus – focus on mesenchymal stem cells
Günter Päth1*, Nikolaos Perakakis2, Christos S. Mantzoros2, Jochen Seufert1
1 Division of Endocrinology and Diabetology, Department of Medicine II, Medical Center –
University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
2 Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

  • Corresponding author
    Günter Päth, PhD
    Division of Endocrinology and Diabetology
    Department of Medicine II
    Medical Center – University of Freiburg
    Faculty of Medicine, University of Freiburg
    Hugstetter Str. 55
    79106 Freiburg
    GERMANY
    Tel.: +49 761 270 73270
    Fax: +49 761 270 33720
    E-mail: guenter.paeth@uniklinik-freiburg.de
    Keywords
    Stem cells, diabetes mellitus, transplantation, embryonic stem cells, induced pluripotent stem
    cells, mesenchymal stromal cells
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    Abbreviations
    ADA, American Diabetes Association; AE, adverse event; α-SMA, alpha smooth muscle
    actin; 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, CCchemokine
    ligand 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 signalregulated
    kinases; 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-stimulated
    insulin secretion; GVHD, graft-versus-host disease; HbA1c, glycated hemoglobin A1c; HGF,
    hepatocyte growth factor; HLA, human leukocyte antigen; HNF1A, hepatocyte nuclear factor
    1α; HOMA, homeostasis model assessment; HSC, hematopoietic stem cells; HSV-Tk, herpes
    simplex virus thymidine kinase; ICAM1, intercellular adhesion molecule 1; ICOS, inducible
    costimulator; 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, induced
    pluripotent stem cells; ISC, insulin screting cell; ISL-1, insulin gene enhancer protein 1; IU,
    international unit; IV, intravenous; MET, mesenchymal to epithelial transition; MHC, major
    histocompatibility complex; MMP, matrix metalloproteases; MMTT, mixed-meal tolerance
    test; 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, nitric
    oxide; 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 and
    duodenal 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-related
    apoptosis inducing ligand; Treg, regulatory T cell; TSG-6, tumour necrosis factor-stimulated
    gene 6; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling;
    UC, umbilical cord; VCAM1, vascular cell adhesion molecule 1; VEGF, vascular endothelial
    growth factor; WHO, world health organisation; WJ, Wharton’s Jelly.
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    Abstract
    Diabetes mellitus type 1 and type 2 have become a global epidemic with dramatically
    increasing incidences. Poorly controlled diabetes is associated with severe life-threatening
    complications. Beside traditional treatment with insulin and oral anti-diabetic drugs, clinicians
    try to improve patient’s care by cell therapies using embryonic stem cells (ESC), induced
    pluripotent stem cells (iPSC) and adult mesenchymal stem cells (MSC). ESC display a
    virtually unlimited plasticity, including the differentiation into insulin producing β-cells, but
    they raise ethical concerns and bear, like iPSC, the risk of tumours. IPSC may further inherit
    somatic mutations and remaining somatic transcriptional memory upon incomplete reprogramming,
    but allow the generation of patient/disease-specific cell lines. MSC avoid such
    issues but have not been successfully differentiated into β-cells. Instead, MSC and their
    pericyte phenotypes outside the bone marrow have been recognized to secrete numerous
    immunomodulatory and tissue regenerative factors. On this account, the term ‘medicinal
    signaling cells’ has been proposed to define the new conception of a ‘drug store’ for injured
    tissues and to stay with the MSC nomenclature. This review presents the biological
    background and the resulting clinical potential and limitations of ESC, iPSC and MSC, and
    summarizes the current status quo of cell therapeutic concepts and trials.
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    Introduction
    Human organs and tissues possess a limited capacity to completely recover their structure and
    function in a number of pathologic conditions and degenerative diseases. This fact initiated
    the multidisciplinary field of regenerative medicine which investigates the potential of stem
    cells (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 and
    loss of insulin producing pancreatic β-cells in diabetes mellitus (DM). The diverse potential of
    embryonic SC (ESC), induced pluripotent SC (iPSC) and adult mesenchymal SC (MSC) has
    been exploited to restore or maintain insulin secretion as well as to investigate patient-specific
    disease aspects. MSC are currently the most investigated cells in DM-related trials while
    clinical testing of ESC has just started. This review summarizes the biological aspects and the
    application strategies for the treatment of DM by stem cell therapy.
    β-cell replacement
    Patients with autoimmune DM type 1 (T1DM) experience a loss of insulin producing
    pancreatic 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 secretion
    from endogenous β-cells and therefore can only partially reduce the risk for the development
    of micro- (i.e. nephropathy, retinopathy) or macrovascular (i.e. coronary artery disease,
    peripheral artery disease, cerebrovascular disease) complications. Additionally, efforts to
    develop effective immunosuppressive treatments to prevent β-cell loss before disease onset
    had limited success so far [1]. Consequently, restoration of endogenous insulin secretion
    represents an important aim to prevent hyper- and hypoglycemia as well as to reduce or avoid
    diabetic complications and the patient’s requirement for self-management of glycemic control
    by exogenous insulin administration.
    Clinical islet transplantation aims to re-establish endogenous insulin secretion and has been
    steadily refined since its beginning in the 1980s [2]. An important step was the ‘Edmonton
    Protocol’ from 1999 which avoids β-cell toxic glucocorticoids by using sirolimus, tacrolimus
    and daclizumab for immunosuppression [3]. Ongoing clinical research improved isolation,
    culture and transplant techniques, and evaluated advanced anti-inflammatory and
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    immunomodulatory interventions [4-6]. As a result, a multicenter analysis with 18 diabetic
    patients receiving 34 islet transplantations showed a graft survival (defined by C-peptide
    concentrations 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 participants
    receiving 75 islet transplantations, successfully improved glycemic control to a median
    glycated hemoglobin A1c (HbA1c) level of 5.6% after 1 and 2 years [8]. Compared to
    standard insulin therapy, islet transplantation more efficiently improved glycemic control and
    progression of retinopathy, and resolved hypoglycemia even in patients with only partially
    remaining graft function [9, 10].
    Transplantation of whole pancreases is an established alternative to islets and both procedures
    display advantages and limitations [11]. The standard procedure of islet infusion into the liver
    is much safer with less complications than pancreas transplantation which is considered a
    major surgery with accordingly enhanced risks for the patient. Thus, pancreas transplantation
    is rarely performed alone and is most commonly combined with a kidney transplantation in
    patients with T1DM and end-stage renal disease. The major obstacle of the less risky islet
    transplantation is the limited graft survival. Insulin independence after islet transplantation
    initially 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 blockade
    of tumour necrosis factor (TNF), to 50% at 5 years [4]. Thereby, at least experienced islet
    transplantation centers have substantially improved long-term graft function towards the
    reported 5 year outcomes in pancreas transplantation; 55% for pancreas alone and after
    kidney, and 72% for pancreas together with kidney [2, 13].
    As with other organ transplantation, there is a great scarcity of human donor material which
    evoked intensive research on the generation of insulin producing β-cells from SC. Whole
    pancreas transplantation strictly depends on high quality organs while generation of insulin
    producing β-cells from SC has the potential to solve the problem of limited availability of
    donor material for islet transplantation. However, the need for immunosuppression reserves
    both pancreas and islet transplantation as a therapeutic choice to a limited patient population
    such as brittle diabetics with life-threatening hypoglycemic events or subjects who anyway
    receive immunosuppression; e.g. because of kidney transplantations for renal failure due to
    diabetic nephropathy.
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    I. ESC
    ESC represent the inner cell mass of the blastocyst and possess a pluripotent differentiation
    capacity. This makes them capable to form all three germ layers (ectoderm, endoderm and
    mesoderm) which subsequently give rise to all cell types of the body. On this account, these
    cells are considered the superior tool for tissue generation but they evoke ethical concerns
    regarding their origin from human embryos. Moreover, they bear a clinical risk since their
    pluripotent nature makes undifferentiated ESC capable to form teratomas and malignant
    teratocarcinomas in vivo. The development of clinically safe differentiation protocols and
    testing 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 transforming
    processes have to mimic complex embryonal organogenesis in vitro. Differentiation protocols
    therefore established a number of factors and inhibitors that modulate molecular pathways in
    an exact sequential timing to resemble the natural development of pancreatic β-cells.
    Generally, human ESC were firstly differentiated into definitive endoderm cells and then
    sequentially 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 earlier
    attempts proved the concept but did not achieve high yields; e.g. a typical early study resulted
    in 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 protocols
    reached 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 iPSC
    lines raise difficulties in reproducibility outside experienced laboratories since stem cell
    differentiation is despite all progress not a routine procedure.
    A major step in differentiation of human ESC towards β-cells is the expression of the
    transcription factors pancreatic and duodenal homeobox 1 (PDX1) and NK6 homeobox 1
    (NKX6.1) which are markers of pancreatic endoderm and endocrine precursor cells. It has
    been shown that comparable human embryonic pancreas tissue from fetal weeks 6-9, which
    contained very few β-cells at that stage, is capable to mature into functional β-cells after
    transplantation into non-obese diabetic mice with severe combined immunodeficiency
    (NOD/SCID mice) [21]. Based on these findings, in vivo maturation of PDX1+/NKX6.1+
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    progenitors into β-cells has been recognized to be more efficient than mimicking this monthslong
    process in vitro [22]. Furthermore, scalable in vitro differentiation of ESC into endocrine
    pancreatic precursor cells is to date more robust and less complicated [23, 24] than generation
    of fully functional β-cell phenotypes by advanced protocols [17, 19] and therefore the
    favoured strategy of the company ViaCyte [25].
    The resulting improvements in differentiation of ESC towards PDX1+/NKX6.1+ pancreatic
    progenitors during the last decade have also reduced the risk of tumour formation in vivo
    which occurred in a typically early study of the company NovoCell (name changed to
    ViaCyte in 2010) with a rate of 7 out of 46 transplanted mice [22]. Since then, ViaCyte has
    steadily optimized the approach to efficient large-scale production and embedded their
    pancreatic endoderm and endocrine progenitor cells into a macroencapsulation device to
    generate immune isolated VC-01 implants [18, 25, 26].
    Macroencapsulation improved graft survival and clinical safety. Although ESC-derived
    progenitors are hypoimmunogenic, transplanted cells are challenged by adaptive immune
    responses such as local inflammation and rejection. In addition, once maturated into insulin
    producing β-cells, graft cells will be attacked by persistent autoreactive T cells in patients
    with T1DM. Several studies have demonstrated that macroencapsulation protects embedded
    cells by isolation from immune responses and thereby avoids rejection and the need for
    immunosuppression [27, 28]. Furthermore, macroencapsulation prevents escape of embedded
    cells into the body. This is an important safety issue since any ESC transplanted in an
    undifferentiated state bears the potential of malignant transformation. In this view,
    subcutaneously transplanted devices could be retrieved and removed easily. Using this
    concept, Viacyte has achieved a milestone by initiating the first-in-man clinical trial to test
    safety and efficacy of their pancreatic endoderm implant VC-01 in patients with T1DM and
    hypoglycemic 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 ESCderived
    PDX1+/NKX6.1+ progenitors in rodents can be comparably recapitulated in human
    subjects. In addition, two of three further trials initiated by Viacyte aim to test a modified
    implantation device with reduced immuno isolation that allows vascularisation of the
    macroencapsulated cells. Provided that outcome of all these trials will prove safety, efficacy
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    and significant long-term graft function, this approach holds the potential to pave the way
    towards clinical β-cell replacement without immunosuppression and independent of the
    limited availability of donor islets.
    II. iPSC
    The ethical criticism related to the use of human pre-implantation embryos for extraction of
    ESC inspired researchers to refine the work of John B. Gurdon. His key study demonstrated
    that enucleated Xenopus laevis eggs that were transplanted with nuclei from differentiated
    intestinal epithelium could, at least in small numbers, develop into living tadpoles [29]. This
    demonstrated for the first time that somatic cell nuclei have the potential to revert into a
    pluripotent state. Decades passed by before this finding gained broad acceptance in the
    scientific community and the progression of this idea culminated in the birth of the first
    mammal, clone sheep Dolly, on July 5, 1996 [30].
    Gurdons basic concept of reprogrammable somatic cells was further developed and one
    decade after Dolly’s birth, Takahashi and Yamanaka induced embryonic-like pluripotency in
    somatic mouse fibroblasts by viral overexpression of the four transcription factors Sox2,
    Oct4, Klf4 and c-Myc [31]. For their groundbreaking discoveries Gurdon and Yamanaka were
    honored by the Noble Prize in 2012 (www.nobelprize.org).
    As a major achievement, iPSC overcome the ethic obstacle of using embryos for harvest of
    ESC. During ongoing research, the original Yamanaka protocol has been diversely modified
    and viral integration was effectively replaced by treatment with recombinant proteins, small
    molecules and microRNAs [32, 33]. Following the principle route of pancreatic development
    as used for ESC, researchers have successfully differentiated iPSC into functional β-cell
    phenotypes [34, 35] and also established scalable production of both endocrine pancreatic
    progenitors and β-cells [20, 36].
    Nevertheless, further refinement of procedures is still an issue and new tools were created for
    the identification of compounds and conditions which enhance yield and functionality of
    generated β-cells. For example, human iPSC expressing the fluorescent reporters Venus and
    mCherry markers under the control of intrinsic neurogenin 3 and insulin promoters have been
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    generated for screening of differentiation efficiency [37]. These cells have served to identify
    an inhibitor of fibroblast growth factor receptor 1 (FGFR1) that, while blocking the early
    development of pancreatic progenitors, promoted the terminal differentiation of pancreatic
    endocrine progenitors into endocrine cells including β-cells.
    However, due to their origin from adult somatic cells, iPSC can inherit somatic mutations and
    incomplete reprogramming can maintain somatic transcriptional memory including cancer
    associated gene activity [38, 39]. These dangers currently do not define them as the first
    choice for clinical use but, more importantly, iPSC enable the successful generation of
    patient-specific cell phenotypes that allow to recapitulate disease processes in vitro and can
    serve as platforms for drug development and testing [40-42]. For example, researchers
    successfully 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 the
    near future, patient-specific cell lines will help to develop disease-related models that
    overcome the obstacle of species differences between human subjects and animal models.
    III. MSC
    For an excellent graphical overview on MSC biology discussed in section III we recommend
    the 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 that
    BM explants form plastic adherent fibroblast-like clonogenic cells with a high replicative
    capacity in vitro and named them colony forming unit-fibroblasts (CFU-F) [44]. Friedenstein
    et al. further figured out that culture expanded CFU-F are capable to differentiate into
    osteoblasts, chondrocytes and adipocytes, and to reconstitute a hematopoietic
    microenvironment after transplantation in irradiated mice [45, 46]. These findings supported
    the pioneering study of Tavassoli and Crosby who demonstrated that autologous BM
    fragments transplanted into extramedullary sites can reconstitute hematopoietic and
    adventitial structures in rats [47]. The observed process started from a developing network of
    proliferating reticular cells and was successively followed by the occurrence of osteoblasts,
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    osteoid 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 of
    multipotent differentiation into mesenchymal lineages. Based on these features, multipotent
    CFU-F were renamed ‘mesenchymal stem cells’ by A. I. Caplan in 1991 [48]. Caplan later
    commented that the term ‘stem cell’ was provocative at that time but justified by ongoing
    research displaying that CFU-F could generate bone, cartilage, fat, muscle and other
    mesodermal phenotypes in vitro [49]. Postnatally, the contribution of BM-MSC to bone
    formation is associated with declining numbers of MSC after birth as indicated by the 10fold
    drop of CFU-F colony numbers in BM obtained from newborn and skeletally developed
    teenaged donors and a steady further decline with aging [50].
    Thus, bone formation was considered a core function of BM-MSC in vivo and their
    osteogenic 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-MSC
    expressing the genetic marker lacZ and then subcutaneously transplanted into
    immunodeficient mice [51]. The MSC monolayers formed osteoblasts, then the scaffold
    became 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 porous
    scaffolds loaded with BM-MSC significantly contributed to bone repair in rodents and in
    patients 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 the
    abluminal surface of endothelial cells. After subcutaneous transplantation, cells sorted for
    CD146 were capable to organize a hematopoietic microenvironment outside the BM. This
    confirmed 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 BM
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    transplantation in cancer patients after chemo-therapy was tested first-in-man in 1995 and was
    clinically successful and safe [56].
    Lineage tracing, using nestin (Nes)- or leptin receptor (LepR) promoter driven expression of
    the fluorescence reporters GFP or tdTomato, was employed to further investigate MSCrelated
    cell fates in the BM [57, 58]. One should keep in mind that characterization of BM
    lineages is complicated since marker expression like that of Nes-GFP is in part variable
    during cellular development and overlapping between distinct phenotypes. Besides Nes and
    LepR gene activation, the in situ localization of MSC in the BM has been mainly defined by
    the differential expression of CD146, CD271, neural/glial antigen 2 (NG2) and alpha smooth
    muscle actin (α-SMA). Resulting findings indicated the existence of three different MSC
    populations within the endosteal niche and the perivascular niches at arterioles and sinusoids.
    These investigations disclosed that Nes+ MSC of the endosteal niche secrete factors or
    express cell surface molecules that regulate quiescence in nearby HSC [59, 60]. Nes+ MSC in
    the 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 be
    divided in rare periarteriolar NG2+ cells with high nestin expression (Nesbright/NG2+) and
    abundant perisinusoidal LepR+ cells with low nestin expression (Nesdim/Lepr+). Deletion of
    Cxcl12 and Scf in Nes+ MSC results in the mobilization of HSC to extramedullary organs and
    a marked reduction of HSCs in the BM [57, 61, 62]. Notably, the secretion of CXCL12 is in
    part regulated by direct innervation of the sympathetic nervous system and modulated by
    circadian rhythms [63].
    The very low numbers of CFU-F in BM of adult human donors points out that BM-MSC are a
    minor 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 the
    major source of adipocytes and osteoblasts in adult mouse BM but most of these cells
    remained undifferentiated to maintain the hematopoietic niche. The underlying molecular
    regulation was unclear until recently Seike et al. found that CAR/LepR+ cells preferentially
    express early B-cell factor (Ebf) 3 and analyzed its function [65]. Deletion of Ebf3 in
    CAR/LepR+ cells severly impaired HSC niche function and BM became osteosclerotic with
    increased bone in aged mice. Additional deletion of Ebf1 further increased niche dysfunction
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    leading 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 of
    osteogenesis.
    Pericyte-MSC outside the BM
    The early view on MSC as BM stroma cells has nowadays completely changed and the
    occasionally used term ‘mesenchymal stromal cell’ became misleading. Instead, it became
    obvious that MSC within the BM are not part of the connective tissue stroma but are forming
    the endosteal and perivascular niches. Most BM-MSC are of perivascular origin [54]. In a
    landmark study, Crisan et al. clearly documented that MSC phenotypes exist outside the BM
    in multiple organs as perivascular pericytes expressing typical BM-MSC markers like CD146,
    NG2 and α-SMA, and being multipotent for osteogenic, chondrogenic, adipogenic and
    myogenic lineages in vitro [66]. Further functional characterization tested whether such
    pericyte-MSC possess the ability of BM-MSC to restore a hematopoietic niche in irradiated
    mice [67]. The study revealed that sorted CD146+ perivascular cells, isolated from human
    adipose tissue, are capable to support the long-term persistence of transplanted human HSC
    while CD146- perivascular cells did not. This observation clearly demonstrated that BM-MSC
    and pericytes expressing the same markers are equivalent in function.
    Various studies have meanwhile demonstrated that MSC phenotypes could be isolated from
    virtually all tissues of the body including fat, muscle, cord blood, Wharton’s jelly, placenta
    and others [68]. This initiates the notion that possibly a unique MSC may exists but it became
    obvious that all these MSC, beside core markers, display differential gene expression profiles
    in a time and tissue-related manner and thereby affect stemness [69]. For example, muscle
    pericytes are not spontaneously osteochondrogenic while cord blood-derived MSC
    phenotypes display the unique capacity to form cartilage spontaneously in vivo. Furthermore,
    there is evidence that also the intrinsic mechanical properties of the extracellular matrix
    influences cell fate decisions in MSC, as softer matrices that mimic muscle are myogenic
    while rigid matrices that mimic collagenous bone are osteogenic [70]. Collectively, this raises
    the notion that mesenchymal stemness of MSC is adapted to or imprinted by tissue
    microenvironment and that MSC from placenta, Wharton’s jelly, umbilical cord blood etc.
    may display the most embryonal-like phenotype [68, 69].
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    Regarding the notion of developmental and tissue-related differences, Chen et al. recently
    proposed the concept of multiple ‘paralogous’ stem-cell niches which are progressively and
    functionally transformed within an individual organism throughout its life span [58]. In their
    view, delineation of distinct cell phenotypes results from complex multiple interchangeable
    events of epithelial to mesenchymal transition (EMT) and reverse mesenchymal to epithelial
    transition (MET). These dynamic processes make it difficult to discern cell identities and to
    define reliable markers. Therefore, the question whether all pericytes give rise to MSC, or in
    the alternative view, pericyte-MSC differ from BM-MSC but may derive from a common
    progenitor, is not finally answered to date [49, 71, 72]. Surely, the answer will be complex
    and limited by the accuracy and composition of available marker sets; ‘true’ pericyte-MSC
    may likely represent a subpopulation among all pericytes.
    In vivo function of pericyte-MSC
    Pericytes have been discovered in the early 1870s by C.J. Ebert and C.M.B. Rouget [73] and
    were named by K.W. Zimmermann describing their contractile nature in 1923 [74]. Later
    characterization specified that pericytes communicate with endothelial cells by both physical
    contact and secreted factors to regulate growth, stability, architecture and blood flow of
    microvessels as well as they are important for the integrity of the blood brain barrier and
    provide clearance and phagocytosis in the brain [75].
    Pericytes attach to the epithelium by their tips and their contractile apparatus consisting of
    microfilaments containing actin, myosin and tropomyosin enables them to regulate the
    capillary diameter or to move along the microvessels [76]. This indicated that perivascular
    pericyte-MSC and their BM counterparts are not static but dynamic and their close proximity
    to the vasculature enables them to readily mobilize and travel in the bloodstream to sites of
    injury. Consistent with this view, pericytes respond to a series of pro-inflammatory stimuli
    and are able to sense different types of tissue trauma signals by their expressed functional
    pattern-recognition receptors and contribute to the onset of innate immune responses by cellcell
    contact and paracrine effectors [73]. Similarly, transplanted BM-MSC home to various
    sites of injury, e.g. stroke [77], pancreatic islet inflammation and diabetic kidney [78, 79] and
    cancer [80]. Once on site, BM-MSC secrete a variety of immunomodulatory, antiinflammatory,
    angiogenic, anti-apoptotic and tissue-regenerative trophic factors [81], and
    fend off invading microbes by secretion of anti-microbial peptide LL36 that kills bacteria
    upon contact [82, 83].
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    Altogether the ability of migration and humoral tissue restoration is a common feature of
    MSC independent of their BM or pericyte origin. A.I. Caplan, who once coined the term
    MSC, has meanwhile suggested to rename these cells ‘medicinal signalling cells’ to more
    accurately reflect the new conceptional view on MSC as a ‘drug store’ for injured tissues in
    vivo and to preserve the MSC nomenclature [84].
    MSC in cancer
    Besides MSC and HSC, the perivascular niche also accommodates tumour SC and its
    microenvironment has been shown to regulate tumour dormancy and growth [85-87].
    Tumours recruit pericytes by e.g. platelet-derived growth factor (PDGF) to maintain their
    tumour vessels [88] and consequently, inhibition of PDGF receptor signalling causes pericyte
    detachment and vessel regression, and diminishes tumour growth in several cancer models
    [89-91]. Tumour cells further interact with the surrounding stroma leading to a chronically
    increased release of inflammatory cytokines and growth factors [92] that has been described
    as a ‘wound that never heals’ [93]. The chronic inflammatory state drives the recruitment of
    responsive cell types including MSC [94, 95] which account for 0.01–1.1% of total cells in
    prostatectomies from human prostate tumours [96].
    It is now understood that MSC interact with tumour cells at various stages of progression but
    it is not finally clear whether their role is tumour promoting or suppressive. Several cancer
    models implicated that MSC promote tumour progression and invasiveness as well as having
    a role in the creation of a metastatic niche at the secondary site [97-100]. In contrast, MSC
    suppressed tumour growth in several cancer models including breast cancer, Kaposi’s
    sarcoma, hepatoma and melanoma [101-104]. Reasons for conflicting findings may result
    from the heterogeneity of tested MSC populations, differences in experimental design and
    varying responses dependent on the stimuli [105].
    However, it seems unlikely that transplanted MSC have a significant role in inducing or
    promoting tumours in human subjects, as their clinical use has been considered safe since
    1995 [56] and clinicians did not notice a tumour risk. In support of this notion, a metaanalysis
    has studied 1012 participants who received MSC for treatment of ischemic stroke,
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    Crohn’s disease, cardiomyopathy, myocardial infarction, graft versus host disease or served as
    healthy volunteers but did not find any indication of malignancy [106].
    Meanwhile, research has employed the recruitment of MSC to tumours in order to target
    malignant diseases with genetically modified MSC that, for example, overexpress pigment
    epithelium-derived factor (PEDF) to reduce angiogenesis or overexpress TNF-related
    apoptosis inducing ligand (TRAIL) to induce apoptosis [107]. Despite using MSC from
    different sources, different transfection methods and a wide array of expressed proteins, the
    data 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 which
    aimed to target advanced gastrointestinal cancer (ClinicalTrials.gov: NCT02008539).
    The trial used ganciclovir (GCV) in combination with autologous BM-MSC overexpressing
    herpes simplex virus thymidine kinase (HSV-Tk) under the control of the CC-chemokine
    ligand 5 (CCL5/RANTES) promoter. Mechanistically, engineered MSC migrate to tumours
    where they become activated to express CCL5 [98]. Subsequently induced HSV-Tk
    phosphorylates GCV which then inhibits DNA polymerases and thereby induces apoptosis in
    transfected 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 found
    generally favorable with stable disease in four patients, and progressive disease in 2 patients
    after one year follow-up [109]. Slowed tumour progression and enhanced survival are of great
    importance in the field and engineered MSC may contribute in the future to prolong the life of
    cancer patients. Side note: the HSV-Tk/GCV suicide gene technique has also been tested as
    an ’emergency switch’ that would allow to eliminate transplanted iPSC in case of malfunction
    [110].
    Transdifferentiation of MSC into insulin producing pancreatic β-cells
    There was initially great optimism that MSC could be easily transdifferentiated across the
    germ layer border into insulin producing pancreatic β-cells and thereby avoid the ethical and
    tumorigenic obstacles of ESC and iPSC. Generation of insulin producing cells from MSC
    employed genetic engineering including overexpression of PDX1, neurogenin 3 (NGN3) and
    paired box 4 (PAX4) [111-113] and/or complex in vitro protocols using various conditions
    and factors to resemble pancreatic development [114-118]. Depletion of β-cells in rodents by
    a high dosage of the β-cell toxic compound streptozotocin (STZ) has frequently been used to
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    test the functional capacity of transplanted insulin producing cells. As a variant related to
    aspects of T2DM [119], multiple low-dose STZ-treatment causes islet inflammation for
    testing of MSC-mediated recovery of β-cell dysfunction and partial loss. Aspects related to
    the immunology of T1DM have been investigated in female NOD mice with spontaneously
    occurring autoimmune insulitis [120].
    Several studies reported that transplanted MSC-derived insulin producing cells can improve
    glycemia in STZ-diabetic rodents [111, 114, 118, 121]. Nevertheless, stem cell specialists
    remained sceptic concerning in vitro transdifferentiation of MSC beyond mesodermal
    lineages. In this regard, the efficiency of MSC transdifferentiation was generally very low and
    resulting insulin producing phenotypes frequently possessed an acurate secretory capacity but
    were not further expandable or vice versa. To date, transdifferentiation of MSC has not
    reached clinically significant large scale production of pancreatic progenitors or β-cells as it
    has been established for ESC and iPSC.
    The study of Ianus et al. initiated the notion that injected BM cells contain a subpopulation of
    cells that engraft into islets and are capable to transdifferentiate into insulin producing
    phenotypes in vivo [122]. It was observed that injection of BM cells with insulin gene 2
    promoter driven GFP expression into sublethally irradiated mice gave rise to a small
    proportion of 1.7-3% glucose-responsive GFP+/insulin+ cells within islets which, after
    isolation 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 BM
    cells from GFP mice in NOD/SCID mice with multiple low-dose STZ-induced islet
    inflammation and noted partial recovery of diabetic blood glucose levels [78]. GFP-BM-cells
    significantly migrate to the inflamed endocrine pancreas and their occurrence within islets
    was associated with enhanced local proliferation and 2.5% GFP+/insulin+ cells. Since the
    insulin+/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 incompletely
    differentiated 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 2
    GFP+/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 than
    transdifferentiation [124, 125]. Importantly, an elaborated lineage tracing study from Douglas
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    Melton’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 study
    reported that up to 3% of injected human BM-MSC engrafted into inflamed pancreatic islets
    of multiple low-dose STZ-diabetic NOD/SCID and improved hyperglycemia by reduction of
    β-cell loss and partly maintained mouse insulin blood levels in the absence of detectable
    human insulin [79]. In addition, up to 11% of injected human BM-MSC engrafted into the
    STZ-injured kidneys and improved glomerular morphology as well as decreased mesangial
    thickening and macrophage infiltration.
    Collectively, these studies exclude significant transdifferentiation of MSC into insulin
    producing cells in vivo and pave the way for the new understanding that MSC migrate to and
    engraft at site of injury to support tissue repair by secretion of numerous tissue regenerative
    factors [81].
    Humoral potential of MSC
    After isolation, pancreatic islets suffer from hypoxic culture stress due to loss of blood supply
    and consequently impeded transport of oxygen to the inner cell layers of the threedimensional
    islet structure [127, 128]. After transplantation, islets were further challenged by
    local inflammation and rejection processes [129]. Fast dynamics of revascularisation and
    downregulation of immune responses have been considered important for long-term graft
    function and generated interest on the angiogenic and immunomodulatory potential of MSC
    in the context of islet transplantation.
    Kinnaird et al. displayed that human MSC express a wide array of arteriogenic cytokine genes
    and that MSC conditioned media promoted smooth muscle cell proliferation and migration in
    a dose-dependent manner in vitro [130]. In vivo, using a murine hindlimb ischemia model,
    murine MSC conditioned media enhanced collateral flow recovery and remodeling, improved
    limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy
    compared with control media. In this regard, Figliuzzi et al. tested the angiogenic effects of
    BM-MSC on co-transplanted islets in STZ-diabetic rats and noted that improved graft
    survival and function in association with increased numbers of new capillaries and expression
    of vascular endothelial growth factor (VEGF) [131]. Upcoming studies confirmed the
    angiogenic capacity of MSC and its association with VEGF [132-135]. In this regard, it was
    shown in vitro that VEGF inhibition partially blocked the enhanced formation of
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    anastomosing tubule networks by co-cultured endothelial cells [134]. Therefore, VEGF
    appears to be an important player which is supported by other MSC-derived factors such as
    nerve growth factor (NGF) [136] and factors inducing angiopoietin receptor Tie-2 expression
    in islets [135]. In sum, these studies established that MSC-mediated revascularisation
    contributes to islet graft survival by shortening the post-transplantation ischemia period.
    Improved revascularization and functional outcome of co-transplanted islet grafts have been
    further associated with reduced numbers of terminal deoxynucleotidyl transferase mediated
    dUTP nick end labeling (TUNEL)+ and caspase-3+ apoptotic cells [137, 138]. Angiogenic
    VEGF and several other MSC-released trophic factors including hepatocyte growth factor
    (HGF), insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β, basic
    fibroblast growth factor (bFGF) and granulocyte-macrophage colony-stimulating factor (GMCSF)
    display anti-apoptotic properties [81, 139]. The potential of MSC to mediate survival
    was tested by direct interactions with β-cells in vitro in the absence of third party cells from
    surrounding tissues.
    In line with the variety of released growth factors, we and others displayed that MSCconditioned
    medium or co-cultured MSC preserve Akt signaling in cultured islets undergoing
    hypoxic culture stress and additional treatment with alloxan and STZ [135, 140]. Akt
    signaling promotes survival and reduces intrinsic apoptosis by its influence on B-cell
    lymphoma 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 extracellular
    signal–regulated kinases (ERK)1/2 signaling which, similar to Akt, promotes survival by
    inhibition of intrinsic apoptosis [142, 143]. Interestingly, MSC induced ERK1/2 signaling
    only in highly proliferative endothelial cells and INS-1E insulinoma cells but not in primary
    mouse islets with a low proliferation rate [135, 140, 144]. These observations indicate an
    important 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-cell
    contacts since humoral improvement of glucose-stimulated insulin secretion (GSIS) in
    indirect co-cultures with cells separated by membranes [135] was not well reproducible by
    other studies unless cells were cultured in direct contact [146, 147]. In this respect, MSC
    enhance GSIS in vitro by release of annexin-1 (ANXA1) while MSC from Anxa1-/- mice had
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    no functional capacity [148]. Hence, heterogeneous effects of MSC on GSIS may partly result
    from different expression levels of ANXA1. Likely, the very close proximity of MSC and
    islets 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 upregulated
    expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule
    1 (VCAM1) [149]. Such supportive processes are an important topic in tissue engineering and
    it has been recognized that islets, which lost ECM during enzymatic isolation, show improved
    survival 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 receptor
    antagonist (IL-1RA) and tumour necrosis factor-stimulated gene 6 (TSG-6) [81, 139, 151]. As
    a result, MSC have been described to induce regulatory T cells and anti-inflammatory M2
    macrophages, and to inhibit T cells, natural killer cells and T helper (Th)17 cell differentiation
    as well as maturation of dendritic cells (DC).
    Consequently, MSC substantially reduced co-transplanted islet graft inflammation and
    rejection in BALB/c mice [138, 152], humanized NOD scid gamma (NSG) mice [153] and a
    cynomolgus monkey model [154]. All these studies showed MSC-improved engraftment in
    association with reduced infiltration of T cells and neutrophils, and increased numbers of
    circulating regulatory T cells. Inhibitors established that MSC-mediated prevention of T cell
    proliferation and islet graft rejection was not related to IDO and heme oxignase-1, partially
    related to NO and profoundly mediated by matrix metalloproteases (MMP)-2 and MMP-9 via
    reduction of IL-2 receptors on T cells [152]. MSC further suppress the proliferation and
    activation of T cells by interaction with IL-10-producing CD14+ monocytes [153].
    Remarkably, systemically injected MSC in female NOD mice reduced the incidence of
    spontaneous T1DM [155] or reversed recent-onset hyperglycemia via release of programmed
    cell 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/Th2
    cytokine balance in blood and induced apoptosis of infiltrated leukocytes in pancreatic islets
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    [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 which
    is a frequent diabetic complication. Endogenous MSC, present in the skin as dermal sheath
    cells surrounding hair follicle units [160] and as perivascular pericytes [161]. Skin injury
    induces MSC to recruit and activate epithelial cells, fibroblasts and keratinocytes to
    revascularize 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 with
    MSC-conditioned medium [164-166]. Wound healing is impaired in DM patients which show
    degraded micro- and macrovessels in association with early occurring detachment and loss of
    vascular pericytes at capillaries [167, 168]. Importantly, MSC-treatment successfully
    improved wound healing under diabetic conditions such as in diabetic db/db mice with
    mutated leptin receptor [169] and a rat model of diabetic foot ulceration [170].
    MSC in clinical trials
    The complex and wide-ranged humoral potential of MSC attracted much attention among
    researchers and clinicians. MSC can be isolated from various tissues, frequently from BM and
    adipose tissue, by minimal invasive puncture and they also allow noninvasive retrieval from
    often discarded ‘medical waste’ such as placenta, cord blood and umbilical cord [68, 171]. It is
    further known from early CFU-F studies and numerous studies since then that MSC could be
    easily expanded in vitro without significant loss of their mesenchymal differentiation capacity
    or their humoral secretion. Moreover, MSC are immune-privileged because they express very
    low levels of major histocompatibility complex (MHC) class I and no MHC class II which
    normally prevents or strongly reduces immune responses [172, 173]. In clinical use since
    1995 [56], MSC are considered clinically safe [106] and both administration of autologous
    and 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 broad
    array of diseases including hematological disease, graft-versus-host disease (GVHD), organ
    transplantation, cardiovascular and neurological diseases, bone and cartilage repair as well as
    inflammatory and autoimmune diseases [177]. Among these, more than 60 trials address
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    T1DM and T2DM, and from these we have summarized all trials with reported outcome in
    Table 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 but
    can not recover unless thereby induced autoimmune responses are blocked [178]. MSC
    mediate 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]. In
    T2DM, the anti-inflammatory features of MSC were used to ameliorate chronic low-grade
    inflammation 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 factors
    should improve engraftment and survival of transplanted islets [181].
    Interestingly, there is only one completed trial investigating the effect of MSC cotransplantation
    on islet graft survival and function. Potentially, there are concerns on the
    additional expense needed for generation, testing and application of clinical-grade MSC since
    established immunosuppression regimes should prevent graft rejection. In this regard, Wang
    et al. tested combined autotransplantation of BM-MSC and islets in chronic pancreatitis
    patients undergoing pancreatectomy without immunosuppression [181]. Patients showed
    reduced insulin requirement in the peritransplantation period, reduced decline of C-peptide
    levels after 6 month and lowered fasting blood glucose levels after 12 month. This suggests
    that co-transplanted MSC reduced loss of islet graft function. Additional studies and longterm
    observations 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 who
    required insulin and/or oral anti-diabetic drugs to control glycemia. Currently the total number
    of investigated patients is relatively small. In total, 276 patients were investigated in small
    groups of 6-22 subjects and 3 studies [159, 174, 182] analyzed groups of 31-45 subjects. The
    enrolled patients show diversity regarding age, BMI and other aspects as well as duration and
    severeness of the disease. In this view, also applied MSC came from different sources
    including BM, adipose tissue and umbilical cord, have been differentially processed and
    applied in different dosages. Though MSC did not cure the disease and despite much
    heterogeneity regarding applied MSC, it is quite astonishing that studies reported varying
    positive aspects of partially improved glycemia. Only two T2DM trials reported on diabetic
    complications. Jiang et al. noted without further details that renal and cardiac functions
    showed varying degrees of improvement [183] and Hu et al. found no rise of diabetic
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    complications in MSC-treated patients while placebo-treated patients displayed higher
    incidences 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 of
    outcome that all trials except one [179] reported reduced requirement for exogenous insulin
    and/or anti-diabetic drugs.
    Two studies addressed the question whether MSC treatment could delay development of
    newly-onset T1DM. Hu et al. reported that both the HbA1c and C-peptide were improved
    compared 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 a
    reduced loss of insulin producing β-cells but was not fully reproduced by Carlsson et al. who
    observed improved C-peptide levels in response to a mixed-meal tolerance test but no changes
    in HbA1c, fasting C-peptide and daily insulin dosages after 1 year [179]. More trials with
    prolonged observation periods are needed to clarify the potential of MSC to delay the
    development of T1DM.
    An interesting aspect is the so-called ‘Stem Cell Educator’ [158, 159]. While studies normally
    apply MSC by intravenous injection, the ‘Stem Cell Educator’ approach routed the patient’s
    blood through a closed-loop system that separates lymphocytes from the whole blood and
    briefly co-cultures them with adherent cord blood-derived MSC before returning them into
    the patient’s circulation. Though MSC are not delivered into the body, their temporary contact
    to patient’s lymphocytes was sufficient to induce immune tolerance which ameliorates the
    disturbed Th1/Th2/Th3 cytokine balance with increased Treg numbers in type 1 diabetic
    patients and decreased CD86+/CD14+ monocytes and reduced markers of inflammation in
    type 2 diabetic patients. As a result, all patients displayed a generally reduced requirement for
    insulin and metformin and improved HbA1c values after 10-12 months follow-up. In addition,
    homeostasis model assessment of insulin resistance (HOMA-IR) demonstrated that insulin
    sensitivity was improved post-treatment in type 2 diabetics. The potential of the ‘Stem Cell
    Educator’ is currently further investigated by 2 further trials (ClinicalTrials.com:
    NCT02624804 and NCT03390231).
    Collectively, the trials show that even heterogeneous MSC populations could be clinically
    effective. The question, however, remains whether clinical outcome could be improved by
    optimized MSC batches since actually no standardized method exists for isolation,
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    characterization, expansion, potency testing or pathogen screening [184-186]. A basic issue is
    the donor heterogeneity which has the potential to dramatically influence therapeutical
    properties of MSC. This includes the question whether autologous MSC are a good choice for
    cell therapy of DM since, for eample, hyperglycemia induces pericyte dysfunction via
    activation of p75 neurotrophin receptor/NF-kB-mediated release of microparticles carrying
    miR-503 from neighbouring endothelial cells [187]. Also subclinical inflammation present in
    subjects with metabolic syndrome and T2DM [119] impairs the vascular stem cell niche and
    leads to MSC dysfunction [188]. MSC batches could be further influenced by isolation and
    expansion since several studies suggested that outgrowth from donor tissue generates less
    heterogeneous cell populations with increased proliferation rates and cell viability than
    isolation by enzymatic tissue digestion [189-191]. Moreover, MSC may lose their functions
    due to increased cellular senescence during longer expansion and passaging since a direct
    positive link between early passages of MSC and clinical outcomes in GVHD has been
    demonstrated [192]. One should finally keep in mind that only a small proportion of
    systemically injected MSC engraft at site of injury while most rapidly embolise in the lungs
    and 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 by
    mesenchymal differentiation (into bone, cartilage and fat), plastic-adherent growth in vitro
    and 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 nonclonal
    stromal cell-enriched populations, often misinterpreted as pure SC fractions, which contain
    varying percentages of true MSC, e.g. depending on donor age [50], and thereby plausibly
    exhibit different clinical effectiveness [196, 197]. This demands a reliable assay, such as the
    CFU-F assay, and careful evaluation of each MSC batch may allow the identification of the
    percentage of stem cells and their multilineage potential in each batch of nonclonal MSC.
    Potentially, efficacy of MSC populations could be further enhanced by selection via
    additional markers such as stromal (STRO)-1, CD146, alkaline phosphatase, CD49a, CD271
    and VCAM1 [197]. Against this background, the US Food and Drug Administration (FDA)
    demands registering of tissue processing facilities which should report on (i) prevention of
    transmitting communicable disease via contaminated tissue, (ii) proper handling and
    processing of tissue and (iii) demonstration of clinical safety and effectiveness of cells,
    especially after extensive manipulation.
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    Conclusion on the current state of stem cell therapy of diabets mellitus
    Stem cell therapy has to deal with a wide array of limitations which are still the subject of
    current 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 and
    current developments of macroencapsulation may provide clinical safe usage of these cells
    that demonstrate otherwise potentials for tumor development. Macroencapsulation prevents
    the escape of embedded cells into the body and subcutaneously transplanted devices could be
    retrieved and removed easily. The outcome of Viacyte’s first-in-man trial of their pancreatic
    endoderm implant VC-01 will clarify whether the use of ESC and iPSC is an option in DM
    therapy in the near future.
    MSC are clinically safe and several trials exist though they are limited in number and
    investigated patients. Currently, MSC-based therapy is no cure but shows a potential to
    ameliorate DM since most studies report decreased requirement of exogenous insulin and/or
    anti-diabetic drugs. In this regard, MSC may be best used with diabetic patients that have
    severe problems in controlling glycemia by conventional therapies; e.g. patients with brittle
    DM. We see potential in optimizing their therapeutical performance by standardizing
    isolation, characterization, selection, expansion, potency testing and pathogen screening.
    However, increased efforts will increase costs and MSC have to prove their value.
    Declarations of interest
    None.
    Author contributions
    All authors have participated in manuscript preparation and have approved the final article.
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    Table 1. Clinical trials with reported outcome using MSC to treat DM.
    Section A. T1DM
    Reference,
    Trial number,
    Country,
    Patient number,
    Duration
    Disease
    Objective
    Patient characteristics Treatment Main outcomes
    Wang et al. 2018
    NCT02384018
    United States
    n = 3
    12 months
    Testing safety and effects of
    BM-MSC and islet
    autotransplantation in
    patients with chronic
    pancreatitis undergoing total
    pancreatectomy
    Aged 26, 29 and 42
    years with chronic
    pancreatitis
    Infusion of 20 ± 2.6 x 106
    BM-MSC together with
    5,107 ± 1,920 islet
    equivalents/kg via the
    portal vein
    101 patients with
    pancreatectomy due to
    chronic pancreatitis who
    received islets alone
    served as historical
    controls
    No AE directly related to MSC
    Insulin requirement lowered in the peritransplantation
    period
    Fasting blood glucose lowered
    Fasting C-peptide with smaller declines
    during months 1-6 (mean C-peptide levels
    comparable to control values at 6 months)
    HbA1c not different
    Improved life quality
    Cai et al. 2016
    NCT01374854
    China
    n = 21
    12 months
    Testing safety and effects of
    combined UC-MSC plus
    autologous BM-MNC without
    immunotherapy on C-peptide
    Aged 18-40 years with
    established T1DM for
    2-16 years
    HbA1c 7.5-10.5%
    Fasting serum C-peptide
    < 0.1 pmol/ml
    Daily insulin requirement
    < 100 IU
    Infusion of UC-MSC (1.1 x
    106/kg) and BM-MNC
    (106.8 x 106/kg) through
    supraselective
    pancreatic artery
    cannulation
    Controls received
    standard care (n = 21)
    MSC-treatment was well tolerated
    1 patient with puncture site bleeding
    1 patient with abdominal pain
    Serum C-Peptide AUC 105.7% increase,
    controls 7.7% decrease
    Serum insulin AUC 49.3% increase, controls
    5.7% decrease
    HbA1c decreased 12.6%, controls increased
    by 1.2%
    Reduced insulin requirement (-29.2%) with
    no change in controls
    Carlsson 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 treatment
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    NCT01068951
    Sweden
    n = 9
    12 months
    autologous BM-MSC in
    treatment of patients with
    recently diagnosed T1DM
    T1DM newly diagnosed
    < 3 weeks before
    enrollment
    MMTT-stimulated serum
    C-peptide ˃ 0.1 nmol/l
    106 autologous BMMSC/
    kg
    Sham controls (n = 9)
    HbA1c, fasting C-peptide and insulin
    requirements not different compared to
    controls
    MMTT-induced C-peptide AUC and peak
    values were preserved/increased by MSC
    Dave et al. 2015
    India
    n = 10
    36 months
    Testing co-infusion of ISC (in
    vitro differentiated from
    adipose tissue-derived MSC)
    together with BM-HSC
    Aged 8-45 years
    IDDM for at least 6
    months
    C-peptide levels < 0.5
    ng/ml
    Infusion into portal
    circulation, thymus and
    subcutaneous tissue
    ISC: mean of 3.34 ml cell
    inoculums with 5.25 x
    104 cells/μl; ISC
    expressed ISL1, PAX6
    and IPF1 with mean Cpeptide
    and insulinsecretion
    of 1.03 ng/ml
    and 17.48 l IU/l after 2 h
    in glucose medium
    HSC: mean of 103.5 mL
    with 2.66 x 106/μL
    No untoward effect of stem cell infusion
    Improved serum C-peptide, Hb1Ac, blood
    sugar levels
    Reduced exogenous insulin requirement
    Patients returned to normal lifestyle and
    unrestricted diet
    Thakkar et al. 2015
    India
    n = 20
    24 months
    Testing co-infusion of ISC (in
    vitro differentiated from
    adipose tissue-derived MSC)
    together with BM HSC
    Comparison of autologous vs.
    allogenic stem cells
    Aged 8-45 years
    Group 1: mean age 20.2
    years, mean disease
    duration 8.1 years
    Group 2: mean age 19.7
    years, mean disease
    duration 7.9 years
    Infusion into portal
    circulation, thymus and
    subcutaneous tissue
    Group 1 received 2.65 ±
    0.8 x 104 autologous
    ISC/kg (n = 10); ISC
    expressed ISL1, PAX6
    and IPF1
    Group 2 received 2.07 ±
    0.67 x 104 allogenic
    ISC/kg (n = 10)
    HSC not reported
    No untoward effect, morbidity (pulmonary
    embolism, sepsis) or mortality caused by
    therapy
    Reduced insulin requirement
    Sustained improvement in serum C-peptide
    and HbA1c
    Hu et al. 2013
    China
    Testing the long-term effects of
    WJ-MSC for newly-onset
    T1DM
    Aged 17.6 ± 8.7 years
    T1DM according to ADA
    criteria for less than 6
    2 IV infusions with a mean
    of 1.5-3.2 × 107 WJMSC
    with an interval of
    No acute or chronic side effects compared to
    control group
    Fasting plasma glucose levels not different
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    n = 15
    24 months
    months
    Fasting C-peptide ≥ 0.3
    ng/ml
    1 month
    Control group received
    saline (n = 14)
    from controls
    Improved HbA1c, fasting C-peptide and
    postprandial blood glucose levels
    Reduced insulin requirement
    Zhao et al. 2012
    NCT01350219
    China, Spain
    n = 12
    10 months
    Testing safety and efficacy of
    Stem Cell Educator therapy
    Aged 15 to 41 years
    with a diabetic history
    of 1 to 21 years
    Group A: with some
    residual pancreatic β-
    cell function (n = 6)
    Group B: without (n = 6)
    Stem Cell Educator
    therapy: patient’s blood
    circulated through a
    closed-loop system that
    separates lymphocytes
    from the whole blood
    and briefly co-cultures
    them with adherent CBMSC
    before returning
    them to the patient’s
    circulation
    No AE, minimal pain from two venipunctures
    Reduced insulin requirement (24 weeks)
    Improved fasting C-peptide levels and
    reduced HbA1C (12 weeks) in groups A
    and B
    Increased in basal and glucose-stimulated Cpeptide
    levels (40 weeks, group B)
    Increased expression of co-stimulating
    molecules CD28 and ICOS, increased
    numbers of Tregs and restored
    Th1/Th2/Th3 cytokine balance (4 weeks,
    groups A and B)
    Sham controls (n = 3) without significant
    changes
    Vanikar et al. 2010
    India
    n = 11
    12 months
    Testing co-infusion of allogenic
    ISC (in vitro differentiated
    from adipose tissue-derived
    MSC) together with BM HSC
    Aged 5-45 years
    IDDM for 1-24-years
    Low serum C-peptide
    levels < 0.5 ng/ml
    Intraportal infusion
    ISC: mean of 1.5 ml with
    2.1 x 103/μL; ISC
    expressed ISL1, PAX6
    and IPF1
    HSC: mean of 96.3 ml
    with 28.1 × 103/μL
    No adverse side effect related to stem cell
    infusion or therapy
    Reduced insulin requirement
    HbA1c decreased
    Serum C-peptide levels increased
    Patients became free of diabetic ketoacidosis
    events
    Section B. T2DM
    Reference,
    Trial number,
    Country,
    Patient number,
    Duration
    Disease
    Objective
    Patient characteristics Treatment Main outcomes
    Bhansali et al. 2017 Comparison of safety and Aged 30-60 years with Infusion of 106 BM-MSC 1 patient with local extravasation of blood
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    NCT01759823
    India
    n = 10
    12 months
    effects of autologous BMMSC
    and BM-MNC in
    reducing on insulin
    requirement
    T2DM ≥ 5 years and
    failure to achieve HbA1c
    ≤ 7.5% while receiving
    triple oral anti-diabetic
    drugs in optimal doses
    along with insulin for the
    last 6 months
    (group I, n = 10) or 109
    BM-MNC (group II, n =
    10) via transfemoral
    route into the celiac
    trunk
    Sham controls were
    infused into the femoral
    artery (group III, n = 10)
    following infusion
    Reduced insulin requirement in BM-MSC and
    BM-MNC groups (6 of 10 patients in both
    BM-MSC and BM-MNC groups, but none in
    the control group achieved the primary end
    point of ≥ 50% reduced insulin requirement)
    Increased 2nd-phase C-peptide response in
    BM-MNC group
    Improvement in insulin sensitivity index with
    increased insulin receptor substrate-1 gene
    expression in ABM-MSC group
    Hu et al. 2016
    China
    n = 31
    36 months
    Testing safety and long-term
    effects of WJ-MSC on
    T2DM
    Aged 18-60 years with
    T2DM according to ADA
    criteria
    2 infusions of 106 WJ-MS
    with an interval of 1
    month (group I) through
    veins in the back of the
    hand
    Controls (group II)
    received normal saline
    (n = 30)
    No serious AE
    No chronic side effects or lingering effects
    Fasting plasma glucose almost unchanged
    Reduced insulin requirement
    Fasting C-peptide improved
    HbA1c and postprandial plasma glucose
    improved by trend after 36 months
    HOMA of β-cell secretory function improved
    HOMA-IR unchanged
    Incidence of diabetic retinopathy, neuropathy
    and nephropathy only increased in controls
    Skyler et al. 2015
    NCT01576328
    United States
    n = 45
    3 months
    Testing safety, tolerability,
    and feasibility of allogeneic
    BM-derived STRO-3-
    selected subset of BMderived
    MPC
    (Rexlemestrocel-L,
    Mesoblast Inc.; product
    expressed MSC markers
    STRO-1 and CD146) in
    T2DM inadequately
    controlled with metformin or
    one additional oral anti-
    Aged < 80 years with
    T2DM for 10.1 ± 6 years
    HbA1c 7.0-10.5%
    Metformin either alone or
    in combination with one
    other oral antidiabetic
    medication (except a
    thiazolidinedione) for at
    least 3 months
    IV infusion of 0.3, 1 or 2 x
    106 BM-derived MPC (n
    = 15 each)
    Sham controls (n = 15)
    No serious acute AE due to infusion
    No serious hypoglycemia events or
    discontinuations due to AE, comparable AE
    in MSC and placebo groups, 1 subject with
    severe abdominal pain in MSC group
    No immunologic responses to MSC
    HbA1c reduced by trend, more pronounced
    in patients with baseline HbA1c ≥ 8%
    Insulin requirement reduced by trend
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    diabetic drug
    Guan et al. 2015
    China
    n = 6
    24-43 months
    Testing safety and effects of
    allogenic UC-MSC
    Aged 27-51 years
    Time from hyperglycemia
    to first infusion was 4-
    157 weeks
    Patients treated with
    insulin and poorly
    controlled blood glucose
    levels and HbA1c
    2 IV infusions of 106 UCMSC/
    kg through the
    cubital vein with an
    interval of 14-17 days
    No safety issues during infusion and the
    long-term monitoring period
    Reduced insulin requirement (significant
    during months 1-6), 3 patients became
    insulin-free for 25 to 43 months
    Insulin-free patients displayed reduced
    HbA1c and increased fasting C-peptide
    during months 1-24
    Relative stable fasting plasma glucose and 2
    h postprandial blood glucose levels
    Liu et al. 2014
    Chinese Clinical Trial
    Register ChiCTRONC-
    10000985
    China
    n = 22
    12 months
    Testing safety and effects of
    treatment with allogenic
    WJ-MSC
    Aged 18–70 years
    T2DM according to ADA
    criteria
    Poor glycemic control with
    recent antidiabetic
    therapies, including
    drugs and/or insulin
    injection for at least 3
    months
    GAD antibody negative
    Fasting blood glucose
    level ≥ 7.0 mmol/L
    HbA1c ≥ 7%
    2 infusions of 106/kg WJMSC
    1st infusion via peripheral
    vein on day 5
    2nd infusion directly
    delivered to the
    pancreas via the splenic
    artery using
    endovascular catheters
    on day 10
    3 patients with fever after operative day
    1 patient with subcutaneous hematoma
    1 patient with nausea, vomiting and
    headache
    Improved HbA1c, fasting C-peptide levels,
    HOMA of β-cell secretory function,
    postprandial blood glucose levels
    Reduced insulin requirement and oral
    hypoglycemic drugs
    Reduced serum levels of IL-1β and IL-6, and
    reduced numbers of CD3+ and CD4+ T
    lymphocyte numbers at 6 months
    Kong et al. 2014
    NCT01413035
    China
    n = 18
    6 months
    Testing safety and effects of
    allogenic UC-MSC
    Aged 23-65 years
    T2DM according to WHO
    criteria
    Patients received insulin
    and oral anti-diabetic
    drugs to control
    3 IV infusions of 1-3 x 106
    UC-MSC/kg with an
    interval of 1 week
    4 patients with slight transient fever
    8 patients respond to treatment (efficacy
    group); these show: reduced fasting and
    postprandial blood glucose levels, and by
    trend increased plasma C-peptide levels
    and Treg numbers
    All patients had a feeling of well-being and
    are more active
    Zhao et al. 2013
    NCT01415726
    United States
    Testing safety and efficacy of
    Stem Cell Educator
    therapy
    Aged 29-68 years with
    long-standing T2DM
    Group A: oral medications
    Stem Cell Educator
    therapy: patient’s blood
    circulated through a
    No AE, mild discomfort during venipunctures
    Improved metabolic control and reduced
    inflammation
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    n = 36
    12 months
    (n = 18)
    Group B: oral medications
  • insulin injections (n =
    11)
    Group C: impaired β-cell
    function with oral
    medications + insulin
    injections (n = 7)
    closed-loop system that
    separates lymphocytes
    from the whole blood
    and briefly co-cultures
    them with adherent CBMSC
    before returning
    them to the patient’s
    circulation
    Reduced HbA1C in groups A and B
    Improved insulin sensitivity (HOMA-IR, 4
    weeks)
    Recovery of fasting C-peptide levels in Group
    C (56 weeks) and HOMA of β-cell secretory
    function/C-peptide (12 weeks)
    Improved serum TGF-β, reduced
    CD86+CD14+ monocytes, no effect on
    Treg numbers and restored Th1/Th2/Th3
    cytokine balance (4 weeks)
    Jiang et al. 2011
    China
    n = 10
    6 months
    Testing safety and effects of
    placenta-derived MSC in
    patients with longer
    duration of disease
    Aged 30-85 years
    Duration of DM ≥ 3 years
    Insulin requirement for
    optimal glycemic control
    of ≥ 0.7 IU/kg/day at
    least for 1 year
    3 IV infusions of 1.22-1.51
    x 106/kg placentaderived
    MSC with an
    interval of 1 month
    No fever, chills, liver damage and other side
    effects
    Insulin and C-peptide levels increased
    HbA1c decreased
    Insulin requirement decreased, 4 of 10
    patients achieved reduction of ˃ 50%
    Improved renal and cardiac function (no
    details)
    Abbreviations. ADA, American Diabetes Association; AE, adverse event; AUC, area under the curve; BM, bone marrow; BMI, body
    mass 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, pancreatic
    and duodenal homeobox 1); IR, insulin resistance; ISC, insulin secreting cell; IU, international unit; IV, intravenous; MNC, mononuclear
    cells; MMTT, mixed-meal tolerance test; MPC, mesenchymal precursor cells; MSC, mesenchymal stem cells; PAX6, paired box
    protein 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.
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    Table 2. Summary and comparison of SC in cell therapy of DM.
    ESC iPSC MSC
    Cell type and origin Embryonic SC
    Inner cell mass of the blastocyst
    Adult somatic cells
    Reprogramming in vitro
    Adult SC
    Endosteal (BM) and perivascular niches (all
    tissues)
    Characteristics Pluripotent
    Generates all germ layers:
    ectoderm, endoderm and
    mesoderm
    Pluripotent
    Generates all germ layers:
    ectoderm, endoderm and
    mesoderm
    Multipotent
    Generates mesenchymal lineages: bone,
    cartilage, fat and muscle
    Maintain HSC niche and hematopoiesis
    Ethical concerns Use of embryos No No
    Differentiation into
    pancreatic β cells
    Yes
    Yes
    Insulin+ cells with limited secretory or
    proliferative capacity (experimental)
    Cell therapeutic options β-cell replacement β-cell replacement
    Patient-specific cell lines
    Secreted factors with immunomodulatory,
    angiogenic and tissue regenerative properties
    Advantages Large-scale production of
    pancreatic endoderm, endocrine
    progenitors and fully functional β-
    cells
    Large-scale production of
    pancreatic endoderm, endocrine
    progenitors and fully functional β-
    cells
    Easy isolation and in vitro expansion
    Low immunogenicity allows allogenic
    transplantation without immunosuppression
    Minimal-invasive application
    Clinical safe and well tolerated
    Limitations Tumorigenic if incompletely
    differentiated
    Tumorigenic if incompletely
    differentiated
    Somatic mutations
    Incomplete reprogramming
    maintains somatic transcriptional
    memory
    ISCT minimal criteria for clinical MSC favor
    expansion of nonclonal stromal cell-enriched
    populations with varying proportions of true SC
    Current clinical protocols are not standardized
    and exhibit potential for improvements
    Only a small proportion of systemically injected
    cells engrafts in injured target tissues
    Status First-in-man trial currently
    investigates clinical safety and
    efficacy of macroencapsulated
    ESC-derived pancreatic
    endoderm
    Macroencapsulation avoids
    Currently not safe enough for
    clinical usage
    Patient-specific cell lines allow
    investigation of disease
    processes in vitro and represent a
    platform for drug testing
    Completed clinical trials collectively report on
    reduced requirement for exogenous insulin
    Greatest benefit for patients with problems in
    controlling glycemia by conventional therapy
    More clinical trials in progress
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    tumorigenicity by preventing the
    escape of embedded cells into the
    body and allows easy graft
    removal if necessary
    Abbreviations. BM, bone marrow; DM, diabetes mellitus; HSC, hematopoietic stem cell; ISCT, International Society for Cell Therapy;
    SC, stem cells.
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    Highlights
    Large-scale production of pancreatic endoderm and β-cells from embryonic stem cells
    First-in-man trial investigates macroencapsulated embryonic stem cell-derived β-cells
    Induced pluripotent stem cells allow patient/disease-specific cell lines
    Mesenchymal stem cells secrete immunomodulatory and tissue regenerative factors
    Transplanted mesenchymal stem cells ameliorate human type 1 and type 2 diabetes

Stem cells in the treatment of diabetes mellitus – focus on mesenchymal stem cells