Under a nonadherent state, bone marrow mesenchymal stem cells can be efficiently induced into functional islet-like cell clusters to normalize hyperglycemia in mice: a control study
© Zhang and Dou; licensee BioMed Central Ltd. 2014
Received: 22 January 2014
Accepted: 8 May 2014
Published: 19 May 2014
Bone marrow mesenchymal stem cells (BMSCs) possess low immunogenicity and immunosuppression as an allograft, can differentiate into insulin-producing cells (IPCs) by in vitro induction, and may be a valuable cell source to regenerate pancreatic islets. However, the very low differentiation efficiency of BMSCs towards IPCs under adherent induction has thus far hindered the clinical exploitation of these cells. The aim of this study is to explore a new way to efficiently induce BMSCs into IPCs and lay the groundwork for their clinical exploitation.
In comparison with adherent induction, BMSCs of human first-trimester abortus (hfBMSCs) under a nonadherent state were induced towards IPCs in noncoated plastic dishes using a three-stage induction procedure developed by the authors. Induction effects were evaluated by statistics of the cell clustering rate of induced cells, and ultrastructural observation, dithizone staining, quantitative polymerase chain reaction and immunofluorescence assay, insulin and c-peptide release under glucose stimulus of cell clusters, as well as transplantation test of the cell clusters in diabetic model mice.
With (6.175 ± 0.263) × 105 cells in 508.5 ± 24.5 cell clusters, (3.303 ± 0.331) × 105 single cells and (9.478 ± 0.208) × 105 total cell count on average, 65.08 ± 2.98% hfBMSCs differentiated into pancreatic islet-like cell clusters after nonadherent induction. With (3.993 ± 0.344) × 105 cells in 332.3 ± 41.6 cell clusters, (5.437 ± 0.434) × 105 single cells and (9.430 ± 0.340) × 105 total cell count on average, 42.37 ± 3.70% hfBMSCs differentiated into pancreatic islet-like cell clusters after adherent induction (P < 0.01, n = 10). The former is significantly higher than the latter. Calculated according to the cell clustering rate and IPC percentage in the cell clusters, 29.80 ± 3.95% hfBMSCs differentiated into IPCs after nonadherent induction and 18.40 ± 2.08% hfBMSCs differentiated into IPCs after adherent induction (P < 0.01, n = 10), the former significantly higher than the latter. The cell clusters expressed a broad gene profile related to pancreatic islet cells, released insulin and c-peptide in a glucose concentration-dependent manner, and normalized hyperglycemia of streptozocin-induced mice for at least 80 days following xenograft. Blood glucose of grafted mice rose again after their graft removed. A series of examination of the grafts showed that transplanted cells produced human insulin in recipients.
Our studies demonstrate that nonadherent induction can greatly promote BMSCs to form pancreatic islet-like cell clusters, thereby improving the differentiation efficiency of BMSCs towards IPCs.
Type 1 diabetes (insulin-dependent diabetes mellitus), with an annual net rise of about 3% in incidence , results from the autoimmune lesion of pancreatic beta-cells. Insulin-dependent diabetes mellitus has been successfully treated by transplantation with pancreatic islets of Langerhans [2, 3], but further application of this therapy is limited by the lack of Langerhans donors and immunological rejection. Fortunately, bone marrow mesenchymal stem cells (BMSCs) not only differentiate into multiple mesodermal and nonmesodermal cell lineages including insulin-producing cells (IPCs) [4–11], but also possess low immunogenicity  and immunosuppression by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination [13–21]. These unique biological properties make BMSCs a promising source for cell replacement of kinds of tissue necrosis and function cell exhaustion, especially insulin-dependent diabetes mellitus.
Under adherent induction, however, BMSCs are not effective to form cell clusters, leading to the very low differentiation efficiency of BMSCs towards IPCs. We hypothesized that nonadherent induction favors the formation of cell clusters, thereby improving the differentiation efficiency of BMSCs towards IPCs. To verify this hypothesis, we induced BMSCs of human first-trimester abortus (hfBMSCs) under a nonadherent state to efficiently differentiate into functional islet-like cell clusters, which corrected hyperglycemia in model mice following xenograft. This study provides a new way to induce BMSCs in vitro into pancreatic islet-like cells to treat insulin-dependent diabetes mellitus.
Preparation of hfBMSCs
Under permission of the patients, a local hospital and the Ethics Committee of Northwest A & F University, hfBMSCs were isolated from long bones of 2-month-old to 3-month-old human abortuses using whole-marrow cell culture and proliferated in α-modified Eagle’s medium (Gibco, Billings, Montana, USA), 10% fetal calf serum (Stemcell Technologies Inc., Vancouver, British Columbia, Canada) and 0.1 mmol/l β-mercaptoethanol (Sigma Loveland, CO, USA). The cells were identified using flow cytometry (Beckman Coulter Inc., Fullerton, California, USA) and CD29, CD44, CD166, CD11a, CD14 and CD34 fluorescence-tagged antibodies (Beckman Coulter Inc.).
In vitro induction of hfBMSCs towards insulin-producing cells
Passage 6 of the cryopreserved hfBMSCs was thawed, and proliferated to passage 8 in α-modified Eagle’s medium, 20% fetal calf serum, and 0.1 mmol/l β-mercaptoethanol. Passage 8 of hfBMSCs underwent acclimation in Dulbecco’s modified Eagle’s medium (DMEM)–high glucose (containing 25 mmol/l glucose; HyClone, Logan, Utah, USA), 10% fetal calf serum, and 0.1 mmol/l β-mercaptoethanol, were digested, were transferred into noncoated plastic dishes (in which hfBMSCs are nonadherent), and were induced using a three-stage induction procedure developed by the authors. This procedure was respectively performed 10 times using hfBMSCs from different abortus (n = 10), with nine replications each and each replication covering one dish containing more than 1 × 106 hfBMSCs, as the nonadherent induction group. The same hfBMSCs were transferred into lysine-coated plastic dishes (in which hfBMSCs are adherent) and induced using the same procedure (n = 10) as the adherent induction group.
In stage one of the procedure, the hfBMSCs were cultured for 6 days to develop nestin-positive cells with DMEM–high glucose plus 10 ng/ml basic fibroblast growth factor (Invitrogen, Carlsbad, California, USA), 10 ng/ml epidermal growth factor (EGF; Chemicon, California, USA), 2% B27 supplement (Stemcell Technologies Inc.), 0.5% bovine serum albumin (BSA; Invitrogen), and 0.1 mmol/l β-mercaptoethanol, with medium changes every 2 days. Stage two differentiated nestin-positive cells into IPCs via a 6-day induction with DMEM–high glucose plus 10 ng/ml EGF, 20 ng/ml Activin A (Sigma), 10 mmol/l nicotinamide (Sigma), 2% B27, 0.5% BSA, and 0.1 mmol/l β-mercaptoethanol, with medium changes every 2 days. Stage three was the maturation of the expected IPCs via a 4-day culture with DMEM–low glucose (containing 5.6 mmol/l glucose; Gibco) plus 10 ng/ml EGF, 10 nmol/l exendin-4 (Sigma), 10 ng/ml betacellulin (Sigma), 2% B27, 0.5% BSA, and 0.1 mmol/l β-mercaptoethanol, with medium changes every 2 days.
Statistics of the cell clustering rate
After each time induction, one dish was picked out randomly, cell clusters and single cells in the dishes were collected respectively, and cells from the cell clusters and the single cells were counted respectively. The cell clustering rate was calculated according to the cell number in the cell clusters divided by the summation of the single cells and cells in the cell clusters (n = 10), and the average cell number of each cell cluster was calculated according to the cell number in the cell clusters divided by the cell cluster number (n = 10).
Ultrastructural observation of endocrine cells in cell clusters
After each time induction, one dish was randomly picked from the nonadherent induction group and the adherent induction group (n = 10). The cell clusters were centrifuged for 10 minutes, fixed in 0.1% glutaraldehyde/2% formaldehyde in 0.1 mol/l cacodylate buffer, transferred to 0.1 mol/l cacodylate buffer, and embedded. Ultrathin sections were prepared, observed and photographed under a transmission electron microscope (H-7650; Hitachi, Japan). Fetal pancreatic islets, newly isolated from the pancreas of human abortus at the age of 20 weeks according to Kover and Moore , were used as the positive control.
After each time induction, one dish was randomly picked from the nonadherent induction group and the adherent induction group and cell clusters were collected respectively (n = 10). The cell clusters, non-induced hfBMSCs (as non-induction control), and some fetal pancreatic islets (as positive control) were stained for 15 minutes with a freshly prepared dithizone (Sigma) working solution at 37°C, washed three times with phosphate-buffered saline (PBS), observed and photographed under an optical microscope.
Fluorescent quantitative reverse transcriptase-polymerase chain reaction assay
Human gene-specific primers and quantitative polymerase chain reaction conditions
Primer sequence (5′ to 3′)
Size of PCR product (base pairs)
Annealing temperature (°C)
To test the expression of nestin, insulin and c-peptide during the induction, cell clusters were sampled individually following each induction stage, respectively fixed in 4% paraformaldehyde for 20 to 30 minutes, triple-washed with PBS, and stained with mouse antibodies against human nestin (Abcam, Cambridge, Massachusetts, UK) and insulin (Invitrogen), and rabbit anti-human c-peptide antibody (Abcam), and then with secondary fluorescent antibodies including fluorescein isothiocyanate-conjugated donkey anti-mouse IgG and Alexa Flour 594-conjugated goat anti-rabbit IgG (Invitrogen). To investigate the expression rate of IPCs and other endocrine cells in cell clusters, the cell clusters from each time induction were respectively digested, subcultured for 24 hours in a lysine-coated 24-well plate before fixation, stained with mouse anti-human insulin and glucagon, rabbit anti-human SST and PP antibodies (Invitrogen), and the relevant IgGs conjugated with fluorescence. Ten nonoverlapping visual fields were randomly chosen from each staining sample to count stained and nonstained cells under microscopy, and they were photographed using an inverted microscope. The proportion of cells immunoreactive to particular antigens was quantified according to the stained cell number divided by the summation number of stained cells and nonstained cells (n = 10).
In vitro insulin and c-peptide release in response to increasing glucose concentrations
After each time induction, cell clusters were sampled from the nonadherent induction group and the adherent induction group respectively, transferred into 12-well culture plates containing a lysine coating for cells to attach, 100 clusters per well, and were precultured in DMEM–low glucose, 10 ng/ml EGF, 2% B27, 0.5% BSA, and 0.1 mmol/l β-mercaptoethanol for 24 hours, washed three times with PBS, and stimulated with 1 ml of either 5, 10, or 25 mmol/l glucose in PBS containing 1% BSA (n = 10). In parallel, 100 fetal pancreatic islets (positive control) and 1.2 × 105 hfBMSCs at passage 8 (equivalent to 100 cell clusters, non-induction control) were treated in the same way with all three glucose levels. The supernatants from all treatments were collected after 30 minutes of stimulation, and insulin and c-peptide were measured via radioimmunoassay with the human insulin-specific RIA kit and the human c-peptide RIA kit (LINCO Research Inc., St. Charles, MO, USA) and a liquid scintillation counter (LS6000; Beckman Co., USA).
Preparation of type 1 diabetic model mice
Approved by the Institutional Animal Care and Use Committee of Northwest A & F University, a total of 35 adult male nude mice (BALB/c nu/nu), provided by the Experiment Animal Centre of the Fourth Military Medical University (Xi’an, China), each with normal blood glucose levels, were administered daily intraperitoneal injections of 50 mg/kg streptozocin (STZ; Sigma) for 5 consecutive days. At 72 hours after the last STZ injection, fasting blood glucose levels were determined using a standard blood glucose meter (Sure Step™ Plus; LifeScan Inc., Milpitas, CA, USA) once a day for 3 consecutive days. If the three fasting blood glucose levels were all higher than 18 mmol/l, the mouse was used as a diabetic model animal for our experiments.
Transplantation of CM-DiI-labeled cell clusters to treat diabetic mice
Cell clusters from the nonadherent induction group and the adherent induction group, and also non-induced hfBMSCs, were incubated individually in a working solution of Cell Tracker–CM-DiI (Molecular Probes, Invitrogen Carlsbad, California, USA) for 5 minutes at 37°C and for another 15 minutes at 4°C according to the manufacturer’s instructions, followed by washing with PBS, and were checked under a fluorescence microscope. The 600 CM-DiI-labeled cell clusters from the nonadherent induction (about 7.2 × 105 cells) were transplanted into the right-side testis of 10 diabetic model mice each as the nonadherent induction group (n = 10). The 600 CM-DiI-labeled cell clusters from the adherent induction were transplanted into the right-side testis of another 10 diabetic model mice each as the adherent induction group (n = 10). CM-DiI-labeled but non-induced 7.2 × 105 hfBMSCs were transplanted into the right-side testis of another six diabetic model mice each as the non-induction control group (n = 6). Blood glucose levels of animals in each of the three treatments were measured once every 4 days using a standard blood glucose meter and the body weight was measured once every 8 days using an electronic balance (Mettler-Toledo Inc., Columbus, OH, USA).
To examine the effects of graft removal on the blood glucose fluctuation and the survival situation of the recipients, and the in situ insulin production of the xenografts, three mice were randomly selected from each animal group and their right testes removed 28 days post transplantation following similar blood glucose and body weight measurements. Histological sections of all testicular grafts were dewaxed and stained with mouse monoclonal antibodies against human insulin and then with fluorescein isothiocyanate-conjugated donkey anti-mouse IgG, and were examined using a fluoroscope.
To evaluate the glucose clearance effects of the transplanted islet-like cell clusters, the intraperitoneal glucose tolerance test was performed on five mice in each of the nonadherent induction group and the adherent induction group after 12 days of glucose level normalization following the transplantation and on five nondiabetic mice (as normal control). Each mouse was intraperitoneally injected with glucose at 2 mg/g body weight under fasting conditions and tested for blood glucose at 0, 30, 60, 90, 120, and 150 minutes thereafter .
Data are treated for significance with Student’s t test or SPSS 12.0 simplified Chinese version one-way analysis of variance where applicable. In all cases, all values are presented as mean ± standard deviation and P < 0.05 was considered significant.
Preparation of hfBMSCs
Differentiation of hfBMSCs into islet-like cell clusters
With (6.175 ± 0.263) × 105 cells in 508.5 ± 24.5 cell clusters, (3.303 ± 0.331) × 105 single cells, (9.478 ± 0.208) × 105 total cell count and 1,215.07 ± 35.38 cells in each cluster on average, 65.08 ± 2.98% hfBMSCs differentiated into pancreatic islet-like cell clusters in the nonadherent induction group (n = 10). With (3.993 ± 0.344) × 105 cells in 332.3 ± 41.6 cell clusters, (5.437 ± 0.434) × 105 single cells, (9.430 ± 0.340) × 105 total cell count and 1,199.36 ± 63.41 cells in each cluster on average, 42.37 ± 3.70% hfBMSCs differentiated into pancreatic islet-like cell clusters in the adherent induction group (n = 10). The former is significantly higher than the latter (P < 0.01, n = 10). However, the total cell count and average cell number of each cell cluster are not significantly different between the nonadherent induction group and the adherent induction group (P > 0.05, n = 10). Under transmission electron microscopy, a large number of endocrine cells (Figure 2I) and secretion granules in cytoplasma were observed in the sections of islet-like cell clusters in the nonadherent induction group (Figure 2J) and the adherent induction group (Figure 2K), which were histologically similar to those in fetal pancreatic islets as a positive control (Figure 2L).
Fluorescent quantitative reverse transcriptase-polymerase chain reaction
Fluorescent quantitative reverse transcriptase-polymerase chain reaction (Figure 3B) showed that the islet-like cell clusters in the nonadherent induction group and the adherent induction group all expressed pdx1, ngn3, pax4, neuroD1, nkx2.2, nkx6.1, PCSK1, insulin, glucagon, SST and PP, similar to the expression profile of fetal pancreatic islets. However, the islet-like cell clusters in the nonadherent induction group and the adherent induction group expressed significantly more Ngn3 and Pax4, and significantly less insulin, glucagon, SST and PP than fetal pancreatic islets (all P < 0.01, n = 10), indicating that the cell clusters from hfBMSCs induced in vitro were not mature enough. The expression of the genes was similar between the nonadherent induction group and the adherent induction group.
In vitro insulin and C-peptide release in response to a glucose challenge
Effects of islet-like cell clusters to treat diabetic mice
Nonadherent induction strategies and principle
BMSCs are a type of cells with strong adhesion tendency. Inducing such cells into cell clusters in lysine-coated plastic dishes is difficult. In this study, hfBMSCs in noncoated plastic dishes (under a nonadherent state) were more efficiently induced into functional pancreatic islet-like cell clusters than those in lysine-coated dishes (under an adherent state) using a three-stage induction procedure. The reason for this difference might be that nonadherent induction in noncoated dishes not only facilitated the contact of hfBMSCs with inducers but also provided a good opportunity for the chemotactic movement, aggregation and clustering of hfBMSCs. Cells in cell clusters, affected by surrounding cells via paracrine, easily coordinated each other and differentiated. Chang and colleagues showed that human BMSCs centrifuged into cell pellets more easily differentiated towards IPCs than those under a monolayer . Studies have shown that cell–cell contact between pancreatic beta-cells is important for maintaining their survival and increasing insulin secretion . In the present study, there were IPCs only in the islet-like cell clusters, whereas single cells in adherent growth did not differentiate towards target cells under the same induction according to their shape and immunofluorescence assay (Figure 4), proving that the environment within the cell clusters was able to promote cell differentiation.
Nestin, a marker of neural stem cells, is also expressed by cells located in the epithelium of the pancreatic primordium. Nestin-positive cells generate the cells of the endocrine and exocrine lineages [25, 26], and nestin-positive stem cells from mouse embryonic stem cells can be induced into insulin-positive and c-peptide-positive cells . The newest research indicated that nestin plays pivotal roles as an intermediate regulator governing both stemness and differentiation of stem cells in the process of their differentiation into IPCs . In this study, we efficiently induced hfBMSCs first into nestin-positive multipotential cell clusters, then into pancreatic islet-like cell clusters and finally into functional islet-like cell clusters (Figures 4 and 6) in noncoated plastic dishes using the three-stage induction procedure. However, the characteristics of nestin-positive cells are still debatable.
Differentiation effects of hfBMSCs after nonadherent induction
In this study, 65.08 ± 2.98% of the hfBMSCs under nonadherent induction differentiated into pancreatic islet-like cell clusters, which is significantly higher than their clustering rate (42.37 ± 3.70%) under adherent induction (P < 0.01, n = 10). The differentiation rate of hfBMSCs towards IPCs is 29.80 ± 3.95% in the nonadherent induction group, which is significantly higher than the differentiation rate (18.40 ± 2.08%) in the adherent induction group (P < 0.01, n = 10), as well as higher than the differentiation rate (10 to 20%) of mouse BMSCs in adherence towards IPCs reported by Tang and colleagues  and the differentiation rate (19.8%) of rat BMSCs in adherence towards IPCs reported by Wu and colleagues . These results indicate that nonadherent induction in noncoated plastic dishes can greatly promote BMSCs to form pancreatic islet-like cell clusters, thereby improving the differentiation rate of BMSCs towards IPCs.
In comparison with the fetal pancreatic islets, the islet-like cell clusters from hfBMSCs expressed more Ngn3 and Pax4, the markers of early stage beta-cell development, and secreted relatively lower insulin, glucagon, SST and PP (all P < 0.01, n = 10), indicating that their differentiation was not full in vitro.
Effects of islet-like cell clusters on mouse diabetic treatment
Although some attempts have been made to treat diabetes in an animal model with induced BMSCs in adherence, the results are not ideal. Wu and colleagues reported that the glucose levels of STZ-induced diabetic rats began decreasing at day 6 after the allograft with 5 × 106 induced rat BMSCs in adherence, and remained below 15 mmol/l from day 12 to day 16 post transplantation but elevated again after day 20 . The research by Hisanaga and colleagues showed that the plasma glucose level of STZ-induced diabetic mice markedly fell after the allograft with 5 × 107 induced mouse BMSCs in adherence and the effect continued for at least 4 weeks . Xie and colleagues ameliorated the diabetic conditions of STZ-treated mice by the transplantation of IPCs derived from BMSCs . In this study, the hyperglycemia of diabetic nude mice was normalized by transplanting 600 pancreatic islet-like cell clusters (about 7.2 × 105 cells) from hfBMSCs. The blood glucose levels of three mice rose again after removal of the grafts while seven mice without removal of the graft maintained normal blood glucose levels for at least 80 days (Figure 7A), indicating that the transplanted cells were responsible for the cure of hyperglycemia. This observation was further confirmed by colabeling of CM-DiI and immunofluorescent antibodies against human insulin in the grafts, which demonstrated that the transplanted cells produced functional human insulin in vivo (Figure 8).
In addition, in this study the islet-like cell clusters were transplanted in the testes of model animals because not only there is a strong blood circulation in the testis but also operation of cell transplantation and withdrawal of the graft aimed at the testis is easier than that aimed at other organs, such as a renal subcapsular graft. The transplanted islet-like cell clusters normalized the blood glucose levels of diabetic model mice 12 days after transplantation (Figure 7A), implying that they might go through a process of adaptation, proliferation, maturation and gradual functioning in vivo.
BMSCs from human first-trimester abortus
We are not advocating carrying out abortions for the single purpose of obtaining human fetal BMSCs, but a human abortus is a source of obtaining a variety of fetal stem cells, especially in some countries with family planning policy or birth-control policy. Certainly, such an approach may be controversial in some other countries. The properties of hfBMSCs range between those of embryonic stem cells and adult stem cells, but hfBMSCs form no tumors in mice  whereas embryonic stem cells induced towards IPCs still form tumors in mice [30, 31]. Thus hfBMSCs are an ideal type of seed cell for research on human tissue engineering and regeneration medicine.
This study demonstrates that nonadherent induction can greatly promote BMSCs to form pancreatic islet-like cell clusters, thereby improving the differentiation efficiency of BMSCs towards IPCs.
bone marrow mesenchymal stem cell
bovine serum albumin
Dulbecco’s modified Eagle’s medium
epidermal growth factor
bone marrow mesenchymal stem cell of human first-trimester abortus
The authors thank support from the National Natural Science Foundation of China (31372490) and all members in the Shaanxi Branch of National Stem Cell Engineering and Technology Centre for their invaluable help, especially Dr Yuankui Chu. They also thank the approval of the Ethics Committee of Northwest A & F University and the Institutional Animal Care and Use Committee of Northwest A & F University.
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