Conversion Of Mouse Embryonic Fibroblast And Bone Marrow Mesenchymal Stem Cells Into Functional Osteoblast By Defined Factors

Bone is a well specialized supporting framework of the body,characterized by its rigidity,hardness,regeneration power and repair.It shields the vital organs of the body,serves as an environment for marrow,and acts as a mineral stock for calcium homeostasis and a reservoir of growth factors and cytokines.It also contributes in acid-base balance.Understanding the role of bone cells is important,not only in the orthopedic field,but also in research field involving bone.Bone cells work in harmony to maintain the stability between bone formation and resorption;mean while remote bone structure and function.Bone has a natural ability to regenerate.However,bone grafts are required when complications are met such as excessive bone loss,which impede bone healing.Therefore,bone grafts are in growing demand worldwide.In such approach,autologous bone grafts are considered the "golden key" for bone repair.Even though,in some cases such as large bone loss,significant donor site morbidity and older patients,this procedure may not be practical or has poor outcomes.In these situations,allogeneic bone grafts may alternatively be used,but they have the potential risks of immune rejection,infection transmission,and the limited number of donors in comparison with the increased clinical needs.Tissue engineering is the use of chemical,biological,and engineering principles for regeneration,restoration or repair of living tissue by utilizing cells,biomaterials,and factors alone or in combination.The mechanism of bone tissue engineering is dynamic and complex.It includes migration and recruitment of osteoprogenitor cells,followed by their proliferation,differentiation,matrix formation,and finally remodeling of the generated tissue.The revolution of tissue engineering has raised the success of bone engineering in vitro.Over the past decades,important progress has been obtained in this field,particularly in cells,the biocompatible and biodegradable scaffolds,and the designs of bioreactors,which improve the in vitro osteogenic priming.In hand with identification and discover of many growth factors that can induce endogenous bone repair and vascular formation.Many pre-clinical trials with optimistic results have been conducted using various animal models.Despite this optimism,a poor solid clinical setting remains due to significant issues such as:optimization of cell sources,selection of biomaterial,in vitro preparation and the route of delivery.Based on the previous scientific background,our main goal was to find an available,biological applicable source for bone regeneration to overcome the limitations in Embryonic stem cells(ESCs)and Induced pluripotent stem cells(iPSCs)technology.Direct reprogramming technique was used to convert the mouse embryonic fibroblast(MEF)into functional osteoblast cells.For this purpose,the efficiency of using the human lim mineralization protein-3(hLMP-3)was examined with different combinations of Yamanaka factors.After selection of the suitable cocktail,an in vitro characterization of the osteoblast produced cells was performed.In addition,the selected cocktail was tested in an in vivo orthotopic animal model.Moreover,some natural inducers(Curcumin and All Trans Retinoic Acid(ATRA))were evaluated to reveal their ability to enhance the osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs)in the culture medium.Finally,the osteogenic potency of the hLMP-3 reprogrammed MEF was tested,in an osteogenic differential medium supplemented with curcumin.To achieve these goals,firstly,the genes of interest were cloned inside a lentivirus expression vector.Secondly,these newly constructed lentivirus vectors containing the genes of interest were used for in vitro reprogramming of MEF to osteoblast.Different combinations of Yamanaka factors were tested with hLMP-3 to find out the best reprogramming cocktail.Afterwards,an in vitro characterization of these cells was performed all-over the reprogramming period.Later,this reprogramming cocktail was used to induce bone formation in an induced uni lateral cortical bone defect in an orthotopic animal model.Our results were obtained by specialized morphological,molecular and functional analyses.Finally,looking forward to improve the osteogenic culture medium,some natural inducers(Curcumin and ATRA)were tested during differentiation of BMSCs.Finally,the positive natural inducer was tested in the culture medium during reprogramming of MEF to osteoblast using hLMP-3 lentivirus vector.Our findings revealed the following points:1.Yamanaka transcription factors(Oct4,Sox2,c-Myc and Klf4)and hLMP-3 were successfully cloned inside the lentivirus expression vector(pGMLVPEl).The transduction efficiency,and the multiplicity of infection(MOI)were tested by transducing sub-confluent MEFs at(P3)with Green Fluorescent Protein(GFP)lentivirus vector.Cells were transduced with varying amounts of Lentivirus GFP reporter with different concentration of polybrene.The best result was obtained at MOI=4,and polybrene concentration=4ng/μL.The Fluorescence-activated cell sorting analysis(FACS)confirmed this result,and revealed an increased fluorescence intensity(98.9%)compared with the non-transduced MEF cells.2.MEF cells were directly reprogramed with different combinations of Yamanaka factors with hLMP-3 to convert them into osteoblast cells.After transduction,cells were cultured in an osteogenic medium for 21 days,and examined for osteoblast formation.hLMP-3 gene expression was confirmed in the newly reprogrammed cells using the reverse transcription PCR(RT-PCR)reaction from the isolated RNA of the reprogrammed cells from different groups.The results demonstrated robust expression of the hLMP-3 all over the study period in comparison to the negative expression of the hLMP-3 in the control group(MEF cells).The expression level changes of the bone gene markers during reprogramming revealed that the best reprogramming cocktail that induced the higher expression of bone markers was(c-Myc-Oct4)in addition to hLMP-3.This cocktail induced the highest level of the bone markers’ m-RNA expression all over the study period.Different assessment tools were used to compare the difference between using hLMP-3 overexpression alone(Transdifferentiation)and the overexpression of the selected cocktail(Direct reprogramming).Both groups showed the morphological changes from the spindle-shaped cells of MEF to be flat,followed by relatively polygonal shape.After 14 days,MEF cultures which have been induced to osteoblast,displayed clusters with high cell density and large amounts of an extracellular matrix(ECM),and finally the characteristic appearance of osteoblast cells(cuboidal shape).The results of the in vitro mineralization assay were consistent with the RT-PCR results.We found that after 21 days post transduction,both Alizarin red staining(ALZ),and Von Kossa staining(VK)showed calcium deposition,and bone nodules formation.The staining intensity of c-Myc,Oct4 with hLMP-3 combination was more significant than the staining intensity of hLMP-3 group at(P<0.05)&(P<0.01)for both ALZ&VK respectively.The last step through the characterization process was the evaluation of osteocalcin(OCN)expression using immunofluorescence technique(IF),and on protein level using western blot analysis(WB).The combination of c-Myc,Oct4 and hLMP-3 induced the expression of OCN more than hLMP-3 alone.Thus,this reprogramming cocktail revealed the strongest capability of inducing osteoblast-like cells among our study groups.3-The reprogramming of MEF with c-Myc,Oct4 and LMP-3 together didn’t pass through any intermediate pluripotency stage;by detection of the Nanog(pluripotency marker)expression on cells using IF technique,and RT-PCR at different time points in comparison with iPSCs.Our reprogramming cocktail revealed negative Nanog expression in contrast to the iPSCs that showed positive Nanog expression.Moreover,a significant decrease in c-Myc and Oct4 expression level was observed in comparison with iPSCs.4-The efficiency of the reprogramming cocktail was tested using an in vivo design to reveal the ability of the reprogrammed cells to induce bone formation in an induced unilateral cortical bone defect in the femur of an orthotopic rat animal model.The rat model was selected to proof that the reprogramming cocktail could induce bone formation in different animal models not only in mice.The results of the in vivo experiment proved the in vitro results and that the reprogramming cocktail could induce bone formation inside the bone defect after transplantation.The radiographic and histological examination revealed complete healing within one-month post-transplantation;while the control group didn’t show any signs of healing with many complications occurred as complete femoral fracture in some rats.5-To enrich the osteogenic culture medium,two natural inducers(curcumin&ATRA)were tested in BMSCs culture.First,BMSCs were isolated from five to six weeks old male BALB/c.mice.An almost homogeneous fibroblastic like cell population was observed after 15 days of culture,with little evidence of round or floating cells.These cells were induced to differentiate and proliferate into osteogenic lineage in vitro.The osteogenic medium was supplemented with 15μM curcumin,or 10"6 mol/L ATRA to study their effect on the osteogenic differentiation of the BMSCs.The morphology of BMSCs changed during the first week in an osteogenic medium from the characteristic spindle fibroblast to the characteristic primary osteoblast morphology(cuboidal appearance)in both control(BMSCs in osteogenic medium),and curcumin groups.In contrast,cells in ATRA group displayed a more elongated shape,and dendrites appear clearly detectable.Both curcumin and control groups showed significant staining intensity in ALZ and VK after 28 days.The mineralization was greater in the curcumin group than the control group by measuring the staining intensity of both ALZ and VK(P<0.05)&(P<0.01)respectively.On the other hand,ATRA group did not show any signs of mineralization after staining.The same results were obtained after staining different groups with VK stain after 28 days.Curcumin group showed numerous areas of black deposits indicating sites of calcium deposition and mineralization.The black stained characteristic bone nodules were higher in the curcumin group than the control group;while the ATRA group did not demonstrate any positive staining results.On the same way,the curcumin group showed significant expression of m-RNA bone markers’ over the control and ATRA groups.In addition,curcumin supplemented osteogenic medium increased OCN expression as detected by IF and WB analysis.On the opposite side,ATRA inhibited the mineralization activity of the BMSCs during osteogenic differentiation.Finally,the curcumin supplementation effect on the osteogenic culture medium was examined on MEF cells after reprogramming with hLMP-3.The curcumin-supplemented group increased the hLMP-3 osteogenic potency in comparison to the non-supplemented hLMP-3 group.This was shown by a significance increase in the m-RNA expression of Runx2 at(P<0.01),BMP-2 and Osx at(P<0.05).