MSC population
Pancreatic differentiation protocol
Analysis of markers expression
Functional assays
References
WJ-MSCs
4 steps:
Step 1 with neuronal conditioned medium (NCM) for 7 days
Step 2 H-DMEM/F12 (25 mM glucose), FBS 2 %, nicotinamide, and B27
Step 3 H-DMEM/F12 (25 mM glucose), FBS 2 %, Nicotinamide, B27, and SCM
Step 4 maturation of insulin secreting islet-like clusters
I. Expression of insulin, Glut-2, Hlxb9, Nkx6.1, nkx2.2, and Pdx1 by RT-PCR.
II. Expression of insulin by IHC, IF staining
In vitro: ICC, RT-PCR, insulin, and C-peptide secretion assay
In vivo: STZ-mouse, Insulin ELISA-Kit, IPGT test, IHC, IF staining
[69]
WJ-MSCs in comparison with BM-MSCs
One-step protocol: The cells were seeded in ultra-low attachment culture plates in DMEMF12, with 17.5 mM glucose, 10 mM nicotinamide, 2 nM activin-A 10 nM exendin-4 100 pM HGF and 10 nM pentagastrin
I. Expression of CD29, CD44, CD59, and CD34, by flow cytometry analysis; II. Expression of hC-peptide, h Gcg, h PDX-1 by ICC; and III. expression of insulin by RIA
In vitro: expression of insulin by RIA expression of C-peptide by flow cytometry analysis of viability by cell counting Kit8 and apoptosis by Annexin V-FITC apoptosis detection kit
[70]
WJ-MSCs
3 steps
Step 1: CMRL1066 medium with 10 % FBS, 1 %penicillin/streptomycin/amphotericin B, 100 ng/mL of β-nerve growth factor, 4 nM activin-A, 10 mM nicotinamide, and 25 ng/mL of epidermal growth factor (EGF) for 7 days
Step 2: new medium DMEM/F12 for 7–10 days.
Step 3: 10 mM nicotinamide insulin/transferrin/selenium and 10 ng/mL of basic fibroblastic growth factor were added to medium for 17 days
I. Expression of C-peptide by ICC and immunogold staining on differentiated line
II. Expression of MafA, Pax4, NeuroD, Isl-1, Nkx2.2, Glut2, and insulin by RT-PCR (differentiated line)
In vitro: ICC, immunogold, SEM, Glucose Challenge Test, C-peptide ELISA kit, RT-PCR
In vivo: diabetic NOD mice, IHC, Intraperitoneal glucose tolerance test
[71]
WJ-MSCs
High-glucose DMEM (H-DMEM) with 0.1 mmol/L β-mercaptoethanol and 10 μg/L bFGF until formed fibroblast-like cell. Then washed with 0.1 mol/L PBS and incubated with H-DMEM supplemented with 10 mmol/L nicotinamide
After gene transfection, WJ-MSC were cultured for 24 h with high-glucose DMEM (H-DMEM, 25 mmol/L glucose) supplemented with 10 % FBS and 10−6 mol/L RA. The medium was changed to H-DMEM with 10 % FBS for 2 days and then to low glucose (L-DMEM) with 10 % FBS, 10 mmol/L nicotinamide and 20 ng/mL EGF for 6 days
I. Expression of insulin and glucagon by IFA
II. Expression of insulin and Pdx1 by RT-PCR
In vitro: ICC, IF, RT-PCR
Dithizone staining
Statistical analysis
Radioimmunoassay for insulin
[72]
Human umbilical cord blood cells (CB-MSCs)
5 steps
Step 1, for 24 h H-DMEM, 25 mmol/L glucose with 10 % FBS and 10−6 mol/L retinoic acid, then the medium was changed to H-DMEM with only 10 % FBS for 2 days
Step 2, L-DMEM, with 10 % FBS, 10 mmol/L nicotinamide and 20 ng/mL EGF for 6 days
Step 3, L-DMEM with 10 % FBS and 10 nmol/L exendin-4 for 6 days. Cellular differentiation was monitored by observation of three-dimensional formation of islet-like cell clusters, the expression of genes related to pancreatic endocrine cell development and insulin production
As a control group, cells were cultured in L-DMEM containing only 10 % FBS
I. Expression of GAPDH, insulin, Pdx1, Pax4, Pax6, Ngn3, Isl-1, Nkx6.1, Nkx2.2 and Glut-2 by RT-PCR.
In vitro: IHC, FCA, RT-PCR.
In vivo: STZ mouse model
HPLC result showed that there was no insulin in the control group, but there was insulin in the IPCs grafted group
[73]
Human umbilical cord cells (UC-MSC)
Three steps protocol with four groups. [The four groups included the MSCs control group, pAdxsi-CMV-PDX1 + induction factor group, vector group + induction factor group, and induction factor group.]
I. Expression of insulin, Pdx1, Ngn3, NKX6.1, and Glut-2 by RT-PCR after differentiation
In vitro: pAdxsi-CMV-PDX1 infection and induction of MSCs. ICC, IFA, RT-PCR, WBA, insulin, and C-peptide secretion detection
[74]
Step 1: undifferentiated MSCs with pAdxsi-CMV-PDX1 for 7 days. Step 2: 2 % FBS/DMEM/F12 medium and supplemented with 100 ng/mL EGF and 2 % B27 for 3 days. Step 3: 10 ng/mL glucagons-like peptide-1 (GLP-1), 10 ng/mL betacellulin, 10 ng/mL hepatocyte growth factor (HGF), 10 mmol/L nicotinamide, 2 % B27, 0.1 mmol/L β-mercaptoethanol for 7 days
II. Expression of insulin, Pdx1 by ICC, IFA after induced and uninduced
Human umbilical cord blood cells (CB-MSC)
Protocol I. 6 steps: Step 1: RPMI (without FBS), activin A (100 ng/mL), and Wnt3a (25 ng/mL) for one day. Step 2: RPMI with 0.2 % v/v FBS and activin A (100 ng/mL) for 2 days. Step 3: RPMI with 2 % v/v FBS, fibroblast growth factor 10 (FGF10; 50 ng/mL), and 3-Keto-N-(aminoethyl-amino-caproyl-dihydrocinnamoyl) (KAAD)-cyclopamine (CYC, 0.25 μM) for 4 days. Step 4: DMEM with 1 % v/v B27 supplement, all-trans retinoic acid (RA, 2 μM), CYC (0.25 μM), and FGF10 (50 ng/mL) for 4 days. Step 5: DMEM with 1 % v/v B27supplement, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-phenylglycine t-butyl ester (DAPT; 1 μM) and exendin-4 (50 ng/mL) for 3 days. Step 6: CMRL with 1 % v/v B27 supplement, exendin-4 (50 ng/mL) for 4 days, insulin-like growth factor (IGF-1; 50 ng/mL), and hepatocyte growth factor (HGF; 50 ng/mL)
Protocol II. Six steps: Step 1: RPMI (without FBS), activin A (100 ng/mL), Wnt3a (25 ng/mL) and PI3K inhibitor 50 μM for one day. Step 2: RPMI with 0.2 % v/v FBS and activin A (100 ng/mL) for 2 days. Step 3:RPMI with 2 % v/v FBS, fibroblast growth factor 10 (FGF10; 50 ng/mL), and keratinocyte growth factor (KGF, 50 ng/mL) for 4 days. Step 4: DMEM with 1 % v/v B27 supplement, all-trans retinoic acid (RA, 2 μM), CYC (0.25 μM), and Nogging (50 ng/mL) for 4 days. Step 5: DMEM with 1 % v/v B27supplement for 3 days. Step 6: CMRL with 1 % v/v B27 supplement, exendin-4 (50 ng/mL), insulin-like growth factor (IGF-1; 50 ng/mL), and hepatocyte growth factor (HGF; 50 ng/mL) for 4 days.
In vitro: IFA, RT-PCR, glucose-stimulated C-peptide (ELISA)
In vivo: NOD-SCID. glucose-stimulated C-peptide (ELISA).
Statistical analysis
[75]
Wharton’s jelly stem cells (WJ-MSCs)
Three steps:
Step 1: for 2 days with SFM-A contained DMEM/F12 (1:1) with 17.5 mM glucose, 1 % BSA Cohn fraction V, fatty acid free, 1 % penicillin/streptomycin/amphoteric B, 1× insulin-transferrin selenium-X (ITS-X; 5 mg/L insulin, 5 mg/L transferrin, 5 mg/L selenium), 4 nM activin A, 1 mM sodium butyrate, and 50 μM 2-mercaptoethanol
Step 2: on the third day, the culture medium was changed to SFM-B, which contains DMEM/F12 (1:1) with 17.5 mM glucose, 1 % BSA, 1 %PSA, ITS-X, and 0.3 mM taurine
Step 3: on the fifth day, for the next 5 days, the cell culture was replaced by SFM-C, which contained DMEM/F12 (1:1) with 17.5 mM glucose, 1.5 % BSA, ITS-X, 1 %PSA, 3 mM taurine, 100 nM (GLP)-1 (amide fragment 7–36), 1 mM nicotinamide, and 1× NEAAs
I. Expression of C-peptide by ICC
II. Expression of Pdx1, Pax4, insulin, GPDH by RT-PCR and real-time PCR
In vitro:
I. Measurement of spontaneous C-peptide secretion
II. Glucose challenge test
In vivo: STZ induction in 6- to 8-week old Sprague Dawley rats
I. Measurement of blood glucose
II. Human nuclei and human C-peptide by IHC
III. Statistical analysis
[76]
Human WJ-MSCs
I. Expression of CD40, CD40L, CD80, and CD86 by IF
II. Expression of Pdx1, human insulin, human glucagon, HLA-I, and HLA-DR by qRT-PCR
In vitro:
I. Lymphocyte proliferation assay by Elisa
In vivo: Ad293-EGFP transfected into the HUMSCs and transplanted in STZ induced rats
I. Measurement of body weight, blood glucose, and serum insulin levels
II. Statistical analysis
[77]
Umbilical cord (UB-MSCs) and cord blood mononuclear cells (CB-MNCs)
Three steps protocol:
Step 1 for 2 days, UB-MSC in high glucose DMEM with 1 % β-mercaptoethanol
Step 2 for 6 days in DMEM containing 100 ng/mL endothelial growth factor, 10 ng/mL basic fibroblast growth factor and 2 % B27
Step 3 for 6 days in H-DMEM (25 mmol/L) containing 20 mmol/L nicotinamide
I. Expression of α-SMA, desmin human insulin and C-peptide by ICC
II. Expression of ALP, OPN, LPL, PPARg, glucagon, somatostatin, insulin, Ngn3, β-actin by RT-PCR
III. Expression of CD31, KDR and CD45, CD29, CD90, CD34 by FC
In vivo: different ratio UB-MSCs and CB-MNCs transplanted in STZ-treated C57/BL6 mice 6–8 weeks old
I. Measurement of blood glucose
II. Intraperitoneal glucose tolerance test
III. Expression of human insulin and nuclei antigen by IHC
IV. Human Alu polymerase chain reaction assay
V. Insulin secretion assay
VI. Statistical analysis
[78]
Subsequently, a comparative study carried out by Wu and colleagues compared the differentiation potential of WJ-MSCs and BM-MSCs toward IPCs. Both cellular types were able to form islet-like clusters on the first day of culture in a medium containing nicotinamide, activin A, HGF, exendin-4, and pentagastrin. Notably, the higher expression levels of PDX1 were assessed in differentiated WJ-MSCs with respect to differentiated BM-MSCs. Likewise, secretion of insulin and C-peptide was comparably higher in the differentiated WJ-MSCs [70].
In a more recent paper, Wang and coworkers provided further data derived from in vitro and in vivo experiments using differentiated human WJ cells to treat diabetes in NOD (nonobese diabetic) mice. After transplantation, IPCs were located in the liver and were able to restore physiological glycemia [71]. Authors’ results suggest that WJ-MSCs possess the ability, both in vitro and in vivo, to differentiate into IPCs and revert hyperglycemia.
Wang and colleagues, after isolation of MSCs from human umbilical cord, induced differentiation through a proprietary protocol. First they performed gene transfection via plasmid DNA (NeuroD1 and GFP under the control of CMV promoter) and then they performed the last phase of differentiation, which was called reprogramming-induced differentiation. The authors reported that during the first induction steps the cells changed their morphology, from fibroblastoid to islet cluster like. Differentiated cells started expressing human insulin and glucagon (differently from control cells) and were also positive to dithizone staining. At the mRNA level, differentiated cells did express both human insulin and human PDX1 genes [72].
In another report, Phuc and colleagues isolated MSCs from cryopreserved human umbilical cord blood (UCB) and performed differentiation toward IPCs. After formation of clusters, some typical pancreatic genes, such as Pdx-1, Ngn3, Isl-1, Pax6, Pax6, Pax4, Glut-2, Insulin, Nk2.2, and Nkx6.1, were detected by RT-PCR [73].
He’s group proved the benefit of PDX1 gene transfection along with the administration of a series of induction factors to UC-MSCs in order to obtain IPCs in vitro. The pancreatic differentiation protocol comprised three steps. In particular, insulin and C-peptide expression, as well as positive dithizone stain, were assessed after the third step of differentiation. In addition, insulin, PDX1, and Nkx6.1 expressions were also confirmed by RT-PCR and western blot analyses in induced MSCs. Interestingly, the expression of such genes was restricted to transfected cells alone, whereas untransfected ones, or cells subjected only to the differentiation protocol, failed in expressing such genes [74].
Prabakar and colleagues investigated the use of CB-MSCs for the treatment of diabetes mellitus through in vitro and in vivo experiments. Subsequently to a pancreatic differentiation protocol, the cells expressed key markers such as PDX1, NKX6.1, and NGN3 by immunofluorescence and RT-PCR, thus confirming that CB-MSCs may be successfully differentiated toward a pancreatic lineage [75].
In a parallel report, Tsai et al. performed differentiation experiments using MSCs from hUC, which were induced with a three steps protocol to obtain IPCs. The features of differentiated cells were assessed by immunocytochemistry, real-time PCR, and ELISA. In vivo experiments were performed by transplanting differentiated cells into the liver of diabetic rats via portal infusion. In vitro data showed that pancreatic β-cell development-related genes (such as PDX1, Pax4, and insulin) were expressed in the differentiated cells. Furthermore, C-peptide release was increased after glucose challenge in vitro. In vivo, human nuclei and C-peptide were detected in the rat livers by immunohistochemistry. In addition, after transplantation of differentiated cells into the diabetic rats, blood sugar level decreased [76].
Wang and coworkers performed in vitro and in vivo experiments aimed to the evaluation of the role of immunomodulatory features of WJ-MSCs for the treatment of diabetes. The authors first investigated the immunological features of WJ-MSCs and their effects on lymphocyte proliferation and interferon (IFN)-γ secretion [77]. WJ-MSCs were transplanted into diabetic rats to investigate whether these cells could engraft and differentiate in vivo into pancreatic β cells, and whether the hyperglycemia of diabetic rats could be improved. In vitro data from the authors confirmed that WJ-MSCs did not stimulate lymphocyte proliferation and did not induce allogeneic or xenogeneic immune responses. In vivo experiments showed that after transplantation in diabetic rats, WJ-MSCs were detectable in both liver and pancreas. Interestingly, albeit no differentiation protocols were performed prior to infusion, there was an improvement of hyperglycemia of the diabetic rats. More importantly, the destruction of pancreatic cells was partly reversed. Therefore, the authors concluded that hyperglycemia improvement could be related to the immunomodulatory effects of WJ-MSCs in vivo, even if further experiments are needed to elucidate the actual mechanism [77].
Xiao and colleagues performed in vivo experiments aimed to investigate whether cotransplantation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and cord blood mononuclear cells (CB-MNCs) could reverse hyperglycemia in type 1 diabetic mice. Authors also aimed to determine the appropriate ratio for cotransplantation [78]. UCMSCs and CB-MNCs were transplanted into type 1 diabetic mice at different ratios and blood glucose concentration was monitored in animals. Histology, immunohistochemistry, and human Alu PCR assays were performed to evaluate for the presence of donor-derived cells and the repair of endogenous islets. In separate experiments, the authors also induced UC-MSCs differentiation toward islet-like cells to determine their differentiation potential. Cotransplantation experiments showed that UC-MSCs and CB-MNCs at a ratio of 1:4 effectively reversed hyperglycemia in diabetic mice. Donor-derived cells were detected into pancreas and kidney of transplanted animals. While cells were able to differentiate in vitro toward islet-like cells, human insulin was not detected in the regenerated pancreases; therefore, suggesting that the mechanism of action of the transplanted cells may involve the reactivation of local pancreatic precursor cells, stimulated by the infused cells [78].
A very recent report from Hu and coworkers showed that WJ-MSCs may be administered to treat type 1 diabetes in human patients, and the treatment is safe and prospectively effective [79]. The authors assessed the long-term effects of the implantation of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) on patients with newly onset T1DM. Patients were randomly divided into two groups, patients in group I were treated with WJ-MSCs and patients in group II were treated with normal saline based on insulin intensive therapy. The long-term follow-up of patients was up to 21 months. The authors reported no acute or chronic side effects in group I compared with group II. Clinical parameters such as HbA1c and C peptide were significantly better in group I patients when compared either to pretherapy values or parallel values from group II patients during the follow-up period. These data suggested that the implantation of WJ-MSCs for the treatment of newly onset T1DM may safe and effective.
5 Conclusions
The cumulative data on umbilical cord-derived mesenchymal stem cells, which were showed above, highlight the extreme cellular plasticity and differentiation capacity of these cells. This results in their ability to be differentiated in vitro into IPCs, which are able to respond to glucose levels variations and improve clinical parameters in experimental models of diabetes. The novel data coming from more recent studies highlight that also without a prior differentiation step toward IPC, both cells from umbilical cord matrix and umbilical cord blood may be able to rescue the diabetic phenotype in in vivo models and in patients. Therefore, as shown in Fig. 28.1, the regenerative medicine approach using WJ-MSCs may be based not only on a classical repopulation model but also on the use of WJ-MSCs as support cells for the organ self-repair. It is probable that the frank immune privilege of undifferentiated WJ-MSCs together with their anti-inflammatory activity may be involved in the rescue of local islet cells and progenitors to reduce the disease progression. Comparative studies have demonstrated that WJ-MSCs can be differentiated better than BM-MSCs toward a mature beta cell phenotype, therefore increasing the prospective usefulness of perinatal stem cells in beta cell replacement therapy. The possibility of umbilical cord cells to be banked in parallel to cord blood units, which is desirable for the future, should render these cells available in high numbers for multiple patients. The co-transplantation experiments of UCB-derived and WJ-derived cells push forward for the need of co-banking these two valuable sources of cells for future uses. The present data will be a strong base to generate more research to better characterize the immune features of these cells and of their differentiated progeny and to increase their engraftment potential and survival in vivo.
