Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

Immunomodulatory effect of human umbilical cord mesenchymal stem cells with interleukin-17 receptor-like molecule overexpression on spleen lymphocytes from mice with trinitrobenzene sulfonic acid-induced colitis

AIM: To investigate the immunomodulatory effect of human umbilical cord mesenchymal stem cells with interleukin-17 receptor-like molecule overexpression (IL-17RLM-hUCMSCs) on the spleen lymphocytes from the mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis for providing optimal seed cells to treat inflammatory bowel disease. METHODS: Mesenchymal stem cells were isolated from human umbilical cord. The IL-17RLM gene was transferred into mesenchymal stem cells by lentivirus vector to establish IL-17RLM-hUCMSCs. The experimental colitis mice were induced by TNBS enema, and the spleen lymphocyte suspension was isolated. The lymphocytes were co-cultured with different concentrations of IL-17RLM-hUCMSCs and hUCMSCs under the stimulation of concanavalin A (ConA) for 72 h. The proliferation of lymphocytes was detected by the methods of CCK8 assay and CFSE labeling with lymphocytes+ConA as positive control. The changes of lymphocyte subsets (Th1, Th2, Th17 and Treg) were examined by flow cytometry.RESULTS: Both hUCMSCs and IL-17RLM-hUCMSCs inhibited T-cell proliferation in vitro co-culture system (P < 0.05). When the ratios of MSCs to lymphocytes ranged from 1:1 to 1:10, the inhibitory rates were in a dose-dependent manner. IL-17RLM-hUCMSCs showed higher inhibitory rate than hUCMSCs within the effective concentration range (P < 0.05). Both hUCMSCs and IL-17RLM-hUCMSCs reduced the proportions of Th1 and Th17 cell subsets and increased Treg cell population of spleen lymphocytes from TNBS-induced colitis mice, and IL-17RLM-hUCMSCs showed a stronger inhibitory effect on Th17 cell subset (P < 0.05). CONCLUSION: IL-17RLM-hUCMSCs remarkably inhibit the proliferation of spleen lymphocytes from TNBS-induced colitis mice in a dose-dependent manner. Meanwhile, they regulate immune balance of T cells and have stronger inhibitory effect on Th17 subset.     

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Correlation between the expression of neuron-specific protein and apoptosis in the process of differentiation from rat bone marrow stromal cells into neuron with BDNF

AIM: To investigate the correlation between the expression of neuron-specific protein and apoptosis in the process of differentiation from rat bone marrow stromal cells into neuron with brain-derived neurotrophic factor (BDNF). METHODS: The 5th passage MSCs were induced by BDNF and 2-mercaptoethanol (β-ME), respectively. At 1 h, 6 h, 12 h and 24 h, nestin, neuron specific enolase (NSE), microtubulease associated protein (MAP)-2 and glail fibrillary acidic protein (GFAP) were detected by Western blotting. Cell cycle and apoptosis were examined by flow cytometry. RESULTS: Nestin and NSE of neuron-like cells induced by BDNF and β-ME were all positive by Western blotting. At 12 h, nestin and NSE turned to negative and apoptosis was detected in β-ME group, nestin and NSE still positive and apoptosis wasn't detected in BDNF group. Till 24 h, nestin and NSE in BDNF group were negative but apoptosis still not detected. Notably, GFAP (glial astrocyte marker) was detected and MAP-2 wasn't detected in the two induced groups. CONCLUSION: The down-expression of neuron-specific protein correspondingly with apoptosis in the process of differentiation from MSCs into neuron with β-ME shows that apoptosis may be one of the causes of induced cell death. BDNF induction was not the cause of apoptosis. Other factors may include for the cell death in the presence of neuron-specific protein expression induced by BDNF.     

Human mesenchymal stem cells differentiate into neuron-like cells with Tanshinone II A

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells with Tanshinone II A.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer to determine the effect of the capacity of proliferation and differentiation of the mesenchymal stem cells with FGF-2. hMSC were induced to differentiate into neurons with DMEM Tanshinone II A. Neuron-specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry.RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD11a, CD14, CD34, CD38, CD45, CD80, CD86. FGF-2 have special effect on low denisity MSCs. Simple methods with Tanshinone II A induced hMSC to exhibit a neuronal phenotype, expressing NSE, NF-M, Nestin at 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION:hMSC can be induced to differentiate into neurons with Tanshinone II A.     

Adipose-derived stem cells differentiate to neuron-like cells in vitro

AIM: We isolated and purified adipose-derived stem cells (ASCs) from adipose tissue in order to study their characters and examine their neuron differentiation capacity in vitro. METHODS: ASCs were isolated and cultured. The morphology of ASCs was observed under microscope and their phenotype was examined by flow cytometry. All-trans retinoic acid (ATRA) was used to induce ASCs into neuron-like cells. RT-PCR was used to detect the expression of nestin. Immunohistochemistry and Western blotting were used to detect the expression of neurofilament (NF) and neuron specific enolase (NSE), respectively. RESULTS: ASCs displayed a fibroblast-like morphology adhering to the culture plate. The cells expressed several surface antigens such as CD29 and CD105, while did not express CD31, CD34, CD45 and HLA-DR. Under suitable conditions, various passages of ASCs all had the capacity of neuron differentiation. They expressed nestin, NF and NSE 10 days after adding ATRA in the culture system. CONCLUSION: ASCs isolated from adipose tissue can differentiate into neuron-like cells in vitro. ASCs may be used as an alternative source of cells for neural disease therapy.     

Efficient production of neuron-like cells from embryonic stem cells induced by astrocyte-conditioned medium in vitro

AIM:To investigate whether efficient production of neuron-like cells from embryonic stem cells (ESCs) can be indued by astrocyte-conditioned medium (ACM) in vitro. METHODS:Based on the 4-/4+ protocol established by Bain, two groups were studied:ATRA group, ATRA with ACM group. ESCs were induced into neuron-like cells by means of three-step differentiation in vitro. The totipotency of ESCs was identified by the observation of cells’ morphology and the formations of teratoma in immunocomprised mice. The cell differentiation was evaluated continuously by detection of the cellular specific markers of neural stem cell, neurons and astrocytes such as nestin, NF-200, NSE and GFAP using immuno-histochemistry assay. RESULTS:(1) The ESC-D3 cells kept the ability of differentiation into cellular derivations of all three primary germ layers after continuous passage culture. (2) The ratio of NF-200 and NSE positive cells in the cells induced by ATRA with ACM was higher than that in the cells induced by ATRA only. (3) Finally, the positive rate of the neuron-like cells was up to 73.5% in the group induced by ATRA with ACM. CONCLUSION:The ESCs are induced into neuron-like cells with high purity and efficiency by ATRA with ACM.     

Effects of IL-6 and IL-11 on differentiation of cord blood CD34+ cells towards megakaryocytes

AIM: To investigate the effects of interleukin-6 (IL-6) and interleukin-11 (IL-11) on differentiation of cord blood CD34+ cells towards megakaryocytes and platelet production in vitro.METHODS: The CD34+ cells from fresh umbilical cord blood samples were cultured in serum-free culture medium with thrombopoietin (TPO) 50 μg/L,IL-3 10 μg/L,stem cell factor (SCF) 50 μg/L as control groups,then 10 μg/L IL-6 or IL-11 or IL-6＋IL-11 respectively was added as treatment groups.Mononuclear cells (MNCs) in cultured cells were detected by cell counter,megakaryocytes (CD41+ cells) and platelets were measured by flow cytometry,respectively.Platelet agglutination after thrombin induced was observed by microscopy and flow cytometry.RESULTS: Compared with the control group,the number of MNCs was not significantly different（P>0.05）,but the numbers of CD41+ cells and platelets were increased significantly (P<0.05) in treatment groups.There were more platelet particles in treatment groups than those in control group by microscopy and the results also showed that the cytoplasmic fragments from the cultures responded to thrombin induction.CONCLUSION: It is concluded that both IL-6 and IL-11 induce the cord blood CD34+ cells to differentiate towards megakaryocytes and produce platelets.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.     

The related gene expression in mesenchymal stem cells differentiating to neuron-like cells

AIM: To analyze neuron-related gene expression before and after mesenchymal stem cells (MSCs) differentiating into neuron-like cells. METHODS: MSCs were induced to neuron-like cells with β-mercaptoethanol. Before inducement and at 8 h after inducement, the total RNA was extracted, then the expression of microtubule-associated protein-2 (MAP-2), growth -associated protein -43 (GAP-43), NSE, nestin and neurofilament (NF) mRNA were detected with RT-PCR. RESULTS: NSE mRNA expressed before and after inducement, MAP-2, GAP-43, nestin and NF mRNA only expressed after inducement. CONCLUSION: The differentiation of MSCs into neuron-like cells may be related to MAP-2, GAP-43, nestin and NF expression.     

Hepatocyte growth factor modified human umbilical cord mesenchymal stem cell therapy promotes remyelination after intracerebral hemorrhage in rats

AIM: To study the effects of neurological improvement and remyelination after intracranial hemorrhage (ICH) in rats by a novel therapeutic strategy with hepatocyte growth factor (HGF) gene transfected human umbilical cord mesenchymal stem cells (hUCMSCs) by lentiviral vector. METHODS: ICH was induced in 60 adult male Sprague-Dawley rats by a stereotactically guided injection of bacterial type IV collagenase into the right internal capsule. Non-modified hUCMSCs, HGF-modified hUCMSCs with lentiviral vector or PBS were administered left intraventricularly 7 d after right internal capsule ICH. All rats underwent modified neurological severity scores for 35 d. Luxol fast blue staining, immunohistological staining and Western blotting assessments for myelin basic protein (MBP) were applied. RESULTS: The ICH rats receiving HGF-modified hUCMSCs demonstrated significant functional recovery, determined by modified neurological severity scores, compared to the other groups from 2 weeks after cell therapy. As indicated by Luxol fast blue staining, the percent area of demyelination was obviously reduced in the HGF-hUCMSC treatment group compared to the PBS control group and hUCMSC-only treatment group at 5 weeks after ICH. The expression of MBP detected by immunohistological staining and Western blotting was significantly higher in HGF-hUCMSCs treated brain than that in other groups (P<0.01). CONCLUSION: Our data suggest that the HGF gene-modified hUCMSCs contribute to the remarkable functional recovery after ICH compared to hUCMSCs transplantation alone. The treatment promotes nerve fiber remyelination by up-regulating the MBP after ICH in rats.     

Isolation and multilineage differentiation of mesenchymal stem cells from human umbilical cord vein in vitro

AIM: To investigate the isolation, purification, expansion and multilineage differentiation of mesenchymal stem cells (MSCs) derived from human umbilical cord vein in vitro.METHODS: By 1% collagenase Ⅱ digestion, endothelial cells were isolated from human umbilical cord vein and cultured in IMDM medium.The morphology of the cells was observed by Wright’s staining and electron microscope.Cell cycle and immunophenotype were investigated by flow cytometry.Assays of adipogenic and osteogenic differentiation were performed in vitro.von Kossa staining, Oil Red O staining and mRNA expression of osteopontin and lipoprotein lipase were studied in the induced cells.RESULTS: The cells from the cord vein displayed a fibroblast-like morphology adhering to the culture plate.FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and HLA-DR were negative.Adipocyte and osteocyte differentiation were induced successfully.CONCLUSION: The morphology, growth characteristics, immunophenotype and pluripotentiality of the MSCs from human umbilical cord vein are similar to the MSCs from bone marrow (BM).They could potentially be an excellent source of MSCs for experiments and clinics.     

Apoptotic mechanism of neuron-like cells differentiated from adult rat mesenchymal stem cells in vitro

AIM: To investigate the mechanism of apoptosis during the process of adult rat mesenchymal stem cells (MSCs) differentiating into neuron-like cells in vitro. METHODS: The MSCs were isolated primarily from adult rats bone marrow by density gradient and then expanded in medium as undifferentiated cells for 3-5 generations. The MSCs were divided into three groups at random. The control group was induced with β-mercaptoethanol (β-ME). Meanwhile, the U0126 group was given β-ME and U0126 (10 μmol/L) added at the beginning of induction. The PMA groups were treated with β-ME and different concentrations of PMA since pre-induction. The effects of U0126 and PMA on the activity of neuron-like cells were observed by MTT. The effects of U0126 and PMA on the expression of neuron specific marker nestin and expression of apoptosis-related protein caspase, Bcl-2, Bax in neuron-like cells were detected by using immunocytochemistry method. TUNEL technique was used to detect apoptosis index. RESULTS: Compared to control group, neuron viability, nestin and Bcl-2 increased and neuron apoptosis decreased in U0126 group (P<0.05). The activity of neuron-like cells, the expression of nestin and Bcl-2 in experiment group treated with 300 nmol/L PMA decreased (P<0.05), while Bax protein expression and apoptosis index increased (P<0.05). CONCLUSION: Both MAPK and PKC signal pathways may be involved in the differentiation of MSCs into neuron-like cells as well as the apoptotic process in differentiated neuron-like cells.     

Differentiation of neurons from mouse induced pluripotent stem cells in vitro

AIM: To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differentiate into neurons in vitro. METHODS: Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form embryoid bodies (EBs). The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture. Seven days later, Pasteur pipette was used to detach the differentiated cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes, and these “fragments” were transferred to culture dishes with neural stem cell medium. Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum (FBS) and RA. The morphological changes of the cells were observed under inverted microscope. The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein (GFAP) and oligodendrocyte marker myelin basic protein (MBP) were detected by immunofluorescence method. The mRNA expression of GFAP, nestin, β3-tubulin, MAP-2 and MBP was detected by RT-RCR. MAP-2 gene sequence was identified. The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry. RESULTS: Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture. The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope. “Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies. After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS, the typical neurite was observed under the microscope. The neurospheres expressed nestin and their differentiated derivatives expressed MAP-2, GFAP and MBP, respectively. RT-PCR analysis and gene sequencing showed that the neurons were induced successfully. The results of flow cytometry demonstrated that 63.93%±1.47% of iPSCs differentiated into NSCs and 21.4%±1.70% of NSCs differentiated into neurons. CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury.     

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than10passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.     

Ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells during their differentiation into neurons and astrocytes

AIM: To observe the ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells (ADSC) differentiating into neurons and astrocytes. METHODS: ADSC were cultured, amplified and induced with β-mercaptoethanol or 3-isobutyl-1-methylxanthine (IBMX). The expression of nestin in the induced cells was determined by the method of immunofluorescence. The ultrastructure of the induced neural stem cells was observed under transmission electron microscope.RESULTS: The expression of nestin reached the peak at 3 h in β-mercaptoethanol group and at 14 d in IBMX group, and the positive rate of the former (86%) was significantly higher than that of the latter (23%). However, the duration of the induction in IBMX group was obviously longer than that in β-mercaptoethanol group. The ultrastructure of the induced neural stem cells in the two groups was similar under the transmission electron microscope, only with some differences in organelles.CONCLUSION: In the process of ADSC differentiating into astrocytes or neurons with the induction of β-mercaptoethanol or IBMX, the different changes of ultrastructure in ADSC-derived neural stem cells occur in some organelles in cytoplasm, not in nucleus.     

Maturity-promoting protocols and mechanisms directing differentiation of human umbilical cord-derived MSCs towards islet-like clusters

[ABSTRACT]AIM: To determine the maturity-promoting protocols for directing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs）towards islet-like clusters (ILCs) expressing prohormone convertase (PC) 1 and PC2 through different combinations of biological products. METHODS: Primary hUC-MSCs were isolated from the whole human umbilical cord by digestion (with collagenase II), filtration and centrifugation, and then were purified by incomplete digestion. The stem cell-specific markers were detected by the methods of flow cytometry, RT-PCR and immunocytofluorescence. The protocols were adopted for inducing the differentiation of hUC-MSCs towards ILCs. Protocol A consisted of IMDM culture medium containing 10 μg/L basic fibroblast growth factor (bFGF), 10 μg/L epidermal growth factor (EGF), 10 mg/L Ginkgo biloba extract (GBE) and 2% fetal calf serum (FCS). Protocol B was based on the protocol A plus 10 μg/L nicotinamide. Protocol C was based on the protocol B plus 10 μg/L hepatocyte growth factor (HGF). Before and after induction, the morphological changes of hUC-MSCs were observed under inverted microscope. The islet-related mRNA and proteins especially PC1 and PC2 were measured by RT-PCR, qPCR and Western blotting. RESULTS: The cell morphology, surface antigen and stem cell-specific markers indicated that hUC-MSCs were successfully isolated and purified from human umbilical cord. hUC-MSCs expressed nestin broadly and Isl1 to a less degree. ILCs induced by protocol A expressed Glut-2 and MafA mRNA. ILCs induced by protocol B expressed Glut-2, MafA, Nkx6.1 and PC2 mRNA. ILCs induced by protocol C expressed Glut-2, MafA, Nkx6.1, PC2, Ngn3, Pdx1, PC1 and insulin mRNA. The expression of PC1 mRNA was only observed in ILCs induced by protocol C. ILCs induced by protocol B and protocol C both expressed PC2 mRNA and the expression in the cells induced by protocol C was significantly higher than that in the cells induced by protocol B. ILCs induced by protocol C expressed PC1 protein. ILCs induced by protocol B and protocol C both expressed ProPC2 protein and PC2 protein, and the former (protocol B) was less than the latter (protocol C) with statistically difference (P<0.01). CONCLUSION: hUC-MSCs possess the potential to differentiate towards the islet progenitor cells and/or islet cells. Compared to protocol A and B, protocol C offers a maturity-promoting method to induce hUC-MSCs differentiation to ILCs with the expression of PC1 and PC2.