diabetes mellitus is one of major diseases causing heavy burden for many people and countries around the world . these chronic metabolic diseases are caused by absolute ( type 1 diabetes ) or relative ( type 2 diabetes ) insulin deficiency leading to hyperglycemia [ 1 , 2 ] . in both diabetes types , the major determinant for the onset of hyperglycemia and the development of overt disease is an inadequate mass of functional -cells . insulin injection does not mimic the precise and dynamic regulation of -cells on glucose homeostasis , leading on the long term to the development of complications , for example , renal failure or blindness . it also causes a so - called diabetic foot syndrome and results in a tenfold increase in the risk of limb amputation . however , the lack of pancreas donors remains a major obstacle [ 1 , 4 ] . in recent years , mesenchymal stem cells ( mscs ) derived from various tissues have become a research  hot - spot  because of their potential use in regenerative medicine [ 1 , 4 , 5 ] . mscs can be cultured in specially defined conditions and their differentiation extends toward the -cell phenotype and the development of insulin producing cells ( ipcs ) . both islet transplantation and islet - like cells could be used in type 1 diabetes , which is caused by autoimmune destruction of pancreatic insulin producing -cells [ 3 , 7 ] . unfortunately , in the case of islet transplantation , there is a need for immunosuppression . for that reason , mscs differentiated into islet - like cells or insulin producing cells ( ipcs ) could be used [ 3 , 8 ] . thanks to that , some serious drawbacks of islet transplantation ( like shortage of organ donors or graft rejection ) mscs can be obtained from a patient 's autologous tissue , for example , from bone marrow , then expanded and differentiated into ipcs and next transplanted into the same patient [ 3 , 8 ] . the purpose of the study was to develop a three - stage differentiation protocol for obtaining insulin producing cells from mscs . several growth factors , nicotinamide , and high glucose concentration in serum - free dmem were used as cell differentiation inducers . human bone marrow stromal cells ( mscs ) were purchased from a commercial source ( allcells ; catalog number msc-001f ) . cells were seeded into 25  cm culture flasks containing dulbecco 's modified eagle 's medium ( dmem ; biochrom ) with 1000  mg / l glucose , 10% fetal bovine serum , 100  u / ml penicillin , and 100  g / ml streptomycin . medium was removed after 48 hours by washing cells with phosphate - buffered saline ( pbs ; biochrom ) followed by media changes every 2 - 3 days . after growing nearly to confluence , cells were passaged 3 times by being detached with 0.25% trypsin - edta ( gibco ) and reseeded at a density of 5  10 of cells per 25  cm culture flask . when the entire surface of the culture flask was covered by cells , the process of bone marrow - derived mscs induction into insulin producing cells ( ipss ) was started . differentiation of mscs was assessed in cultures of the third passage using a three - stage protocol . the first step included cells ' incubation ( 37c , 5% co2 ) in serum - free high glucose ( 4500  mg / l ) dmem with 0.5  mmol / l -mercaptoethanol ( sigma - aldrich ) for 2 days . in the second step , cells were cultured for 8 days in serum - free high glucose dmem containing 1% nonessential amino acids ( gibco ) , -fibroblast growth factor ( bfgf ) ( at a concentration of 20  ng / ml , 40  ng / ml , 60  ng / ml , or 100  ng / ml ) ( sigma - aldrich ) , epidermal growth factor ( egf ) ( at a concentration of 20  ng / ml , 40  ng / m , 60  ng / ml , or 100  ng / ml ) ( sigma - aldrich ) , and 2% b27 ( gibco ) . in the third step , cells were cultured for additional 8 days in serum - free high glucose dmem containing -cellulin ( sigma - aldrich ) and activin a ( sigma - aldrich ) at a concentration of 10  ng / ml , 20  ng / ml , 30  ng / ml or 50  ng / ml , 2% b27 ( gibco ) , and 10  mmol / l nicotinamide ( sigma - aldrich ) . we used a solution of 0.25% trypsin - edta ( gibco ) for 5 minutes at 37c to detach the cells . next cells were suspended in pbs and washed by centrifugation at 1000  rpm for 5 minutes . then cells were fixed and permeabilized with cytofix / cytoperm solution and perm / wash buffer ( bd pharmingen ) , and the nonspecific epitopes were blocked by incubation in normal goat serum ( santa cruz biotechnology ) . cells were then incubated ( 20  min at room temperature ) with 1  g monoclonal mouse anti - human insulin antibody ( primary antibody recommended for the detection of proinsulin c - peptide of human origin ; santa cruz biotechnology ) . cells treated with primary antibodies were exposed to secondary antibody solution , goat anti - mouse monoclonal igg1 fitc ( santa cruz biotechnology ) . additionally , in 6 of 16 samples cells treated with primary antibody were exposed to fitc - conjugated goat anti - mouse igg f(ab)2 . the results obtained by those secondary antibodies ( isotype specific antibody and igg f(ab)2 ) were concordant ( figure 1 ) . the percentage of positive cells was measured from a cut - off point set using isotype - matched nonspecific control antibody . ten mg of diphenylthiocarbazone ( sigma - aldrich ) was dissolved in 10  ml of dimethyl sulfoxide ( dmso , sigma - aldrich ) . mean values between the two groups were compared using the t - test for independent samples . the expression of proinsulin c - peptide from differentiated ipcs was analyzed by flow cytometry ( figures 1(a)1(c ) ) . we differentiated 16 mscs cultures into ipcs , using different growth factors concentrations , with respect to the concentration used in the protocol . in three cultures we used -fgf and egf at a concentration of 20  ng / ml and activin a and -cellulin at a concentration of 10 ( 1x ) . in another three cultures we increased the concentration of growth factors twice ( 2x ) . in seven cultures the concentration was increased three times ( 3x ) and in three cultures the concentration was increased five times ( 5x ) . flow cytometry analysis of mscs at 18th day showed that increasing concentrations of growth factors positively affected the ability of mscs to differentiate into ipcs . at a concentration of 20  ng / ml of -fgf / egf and 10  ng / ml of activin a and -cellulin the 3.73  1.21% of mscs differentiated into ipcs positive for c - peptide . that means only about 4% of mscs responded to such levels of growth factors , differentiated to -like cells and started to produce insulin . at a concentration of growth factors increased to 40  ng / ml for -fgf / egf and to 20  ng / ml for activin a and -cellulin ( 2x ) 6.59  1.35% ipcs showed c - peptide expression . the highest expression of c - peptide ( 31.4  19.05% ) in ipcs was found at a concentration of growth factor increased three times . nevertheless , at a concentration of growth factors increased five times the percentage of cells positive for c - peptide significantly decreased ( 13.00  2.29% ) ( figure 2 ) . we found that the positive expression of c - peptide was 1.3  0.5% in negative controls ( unstimulated / undifferentiated mscs ) . in flow the samples treated with primary antibody were next exposed to fitc - conjugated goat anti - mouse igg f(ab)2 or goat anti - mouse monoclonal igg1 fitc . there was no significant difference between the two staining protocols ( p = 0.349 ) . when observed under an inversed microscope , undifferentiated mscs looked like long spindle - shaped fibroblastic cells and cultures showed a confluency ( 1st day ; figure 3 ) . some cells did not change their morphology during 18 days of differentiation , but many of them migrated ( 8th day ; figure 3 ) . cells accumulated together and formed spherical islet - like structures , creating aggregated colonies from 10th day and continued to 18th day ( figure 3 ) . to verify the insulin production by the islet - like cells we used diphenylthiocarbazone ( dithizone ) staining . red or strong brown staining indicates insulin positivity in aggregated cells ( figure 4 ) . type 1 diabetes is characterized by destruction of pancreatic -cells , which is the result of autoimmune response . so far , the most commonly used treatment is insulin application , but it has its disadvantages . however , its clinical usefulness has been limited by immune rejection of transplanted islets and the lack of donor islets cells . it has been suggested that regeneration therapy represents a strategy to overcome the challenges in islet transplantation . mscs are promoted as an appropriate population in differentiation of insulin producing cells ( ipcs ) for autologous transplantation . ipcs derived from mesenchymal stem cells could be applied as a cell suspension or they could be used with injectable scaffold . mscs can be isolated from several organs and tissues such as bone marrow , dental pulp , adipose tissues , and umbilical cords . bone marrow derived mscs have some significant advantages such as their high number and relatively easy isolation . mscs are able to differentiate into islet - like cells and possess immunomodulatory abilities , thanks to which it is possible to reduce the risk of immune rejection . indirect differentiation uses chemicals ( e.g. , nicotinamide , growth factors ) as inductors . in this method the use of high glucose concentration in the medium is required , since it is a potent inducer of differentiation . the other method , direct differentiation , is based on a modification of genetic material using , for example , viral vectors . they isolated bone marrow mscs and islets from lewis rats and used a pulse generator for cell electrofusion . it seems that high glucose concentration is a major factor during the process of successful differentiation of mscs into ipcs . tsai et al .   derived ipcs from bone marrow mscs using serum - free high glucose dmem with 1% dmso during the first three - day stage and high glucose dmem supplemented with 10% fbs during 14 days of the second stage . tang et al .   obtained ipcsusing high concentrations of glucose ( 17.5  mmol / l ) in rpmi 1640 medium supplemented with 10% fcs . by culturing the cells in rpmi 1640 medium containing 1  ng / ml fgf , 10  ng / ml egf , and 10  ng / ml hgf for 2 months they promoted cellular differentiation . next , cells were cultured for 7 days in medium containing 5% fcs , 5.5  mmol / l glucose , and 10  mmol / l nicotinamide and 10  nmol / l of exendin 4 . as we can see ipcs the time required for differentiation also varies depending on the reagents used . but according to el - badri and ghoneim   only 5% of mesenchymal stem cells derived from the bone marrow differentiate into ipcs . in the case of msc from other sources , we used indirect differentiation using high glucose concentration ( 4.5  g / l , 25  mmol / l ) among other inducers mentioned above . it is known that glucose promotes cell replication and increases insulin content in the cell . in the current study , differentiating cells formed cell clusters some of which resembled pancreatic islet - like cells . using dithizone we confirmed the presence of insulin in these cells . what is more , the expression of proinsulin c - peptide in differentiated ipcs was analyzed by flow cytometry . our data indicate that , in most cases , we obtained more than 5% insulin producing cells mentioned by el - badri and ghoneim . in the present study , the highest ( mean 31.4 % ) number of c - peptide positive cells was found in cultures stimulated with growth factors at a concentration of 60  ng / ml and 30  ng / ml for -fgf / egf and activin a/-cellulin respectively . we noticed that the increased percentages of differentiated stem cells were associated with higher concentrations of growth factors . we concluded that the concentration of growth factors added to the medium is crucial for the efficiency of ipcs differentiation . on the other hand , we noticed that an increase in the concentration of growth factors above a certain level did not increase the percentage of differentiated cells . our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells . as it is mentioned in the literature , we need about 1  10 of islets for proper pancreatic islets transplantation . our cell culture contained about 1  10  mscs , so taking into account the highest achieved yield ( approximately 30% of functioning -like cells ) , there were about 3  10 of ipcs . calculation shows that we need 50 such cell cultures to obtain the desired results , which is not a big number . some research teams differentiated mscs into ipcs , using different methods , but , after clinical application , the results were unsatisfactory . as we can see , only inconsiderable changes that we made during differentiation resulted in significant enhancement of differentiation levels . the results of our investigation show high level of differentiation of mscs into -like cells , so these findings could be used to clinical applications . obviously , in the future , good manufacturing practice guidelines should provide guidance for manufacturing , testing , and quality assurance in order to ensure that new ipcs can be safe for human consumption . further studies in diabetic mice with higher quality scores can help find more precise results than in vitro studies . at this point , definitely the translation of in vitro research findings to useful clinical applications is a major challenge .
type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing -cells and so far it can be cured only by insulin injection , by pancreas transplantation , or by pancreatic islet cells ' transplantation . 
 the methods are , however , imperfect and have a lot of disadvantages . therefore new solutions are needed . 
 the best one would be the use of differentiated mesenchymal stem cells ( mscs ) . in the present study 
 , we investigated the potential of the bone marrow - derived mscs line for in vitro differentiation into insulin producing cells ( ipss ) . 
 we applied an 18-day protocol to differentiate mscs . 
 differentiating cells formed cell clusters some of which resembled pancreatic islet - like cells . using dithizone 
 we confirmed the presence of insulin in the cells . 
 what is more , the expression of proinsulin c - peptide in differentiated ipcs was analyzed by flow cytometry . 
 for the first time , we investigated the influence of growth factors ' concentration on ipcs differentiation efficiency . we have found that an increase in the concentration of growth factors up to 60  ng / ml of -fgf / egf and 30  ng / ml of activin a/-cellulin increases the percentage of ipcs 
 . further increase of growth factors does not show any increase of the percentage of differentiated cells . 
 our findings suggest that the presented protocol can be adapted for 
 differentiation of insulin producing cells from stem cells .