Mammalian digit/limb as a multi-tissue and complicated organ with a rareregenerative potential is a very challenging issue of regenerative medicine. Combining advancedapproaches such as tissue engineering, transgenic tools, and signaling pathway manipulations maynotably result in functional limb regeneration. In this regard, Shh has particularly marked roles inlimb patterning, and its knockout causes malformation in the limb. On the other hand, theformation of transgenic cells using a safe and effective method such as the non-integrativelentiviral virus (NILV) technique is crucial for human-safe gene therapy. In the present study, weinvestigated the expression of the Shh gene in human bone marrow-derived mesenchymal stemcells (hBMSCs) to produce novel transduced hBMSCs for future application in limb regeneration.Materials and Methods: hBMSCs were achieved from Royan cell bank and identified byassessments of expression of cell surface markers (CD۹۰, CD۱۰۵, CD۷۳, CD۳۴ and CD۴۵) byflow cytometry and also osteogenic/adipose/chondrogenic differentiation. ۳-passages hBMSCswere used for further experiments. Subsequently, to transduction of hBMSCs, the Shh gene wascloned using the NILV method into IRES-GFP viral plasmid to form a Shh-IRES-GFP vector. Toproduce lentiviral particles, the HEK۲۹۳ cells were cultured in a DMEM medium. Next, cells weretransfected by using TurboFect along with the vector and lentiviral packaging vector. Thetransfection medium was renewed after several hours. The filtered viral supernatant was used forhBMSC transduction along with Polybrene (۶ μg/ml). Thereafter, transduced cells were expandedfor several passages in the culture medium. Then, florescent microscopic observation and Realtime PCR were used to evaluate Shh expression in Cells. To investigate the expression of keydownstream genes of shh (MSX۱, Bmp۲, and Fgf۸) RT-PCR analysis was applied. Finally,Histological and Immunocytological (ICC) analysis was used to confirm the gene expression.Results: A large population of cells, were expressed the GFP as a reporter gene that confirmedshh gene transduction. The cells were then sorted and expanded to achieve pure GFP+ cells (SHHhBMSCs.The results of Real-time PCR analysis confirmed that Shh gene was significantlyexpressed into SHH-hBMSCs in comparison to hBMSCs as a control group (***P<۰.۰۰۱). In addition, the expression of SHH and downstream genes such as MSX۱, Bmp۲, and Fgf۸ wasconfirmed by ICC.Conclusion: In summary, our results provide evidences on the Shh expression in hBMSCstransduced using NILV technique. In addition, BMP۲ and FGF۸ as the crucial signaling pathwaysin limb development and regeneration expressed following SHH expression. BMP۲ and FGF۸genes induced osteogenesis and cell proliferation, respectively which are key elements of limbregeneration. So, SHH-hBMSCs are appropriate transduced cells for promoting regeneration inamputated human digit tips after verified in animal models.