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