The Migration And Homing Of Hair Follicle-derived Mesenchymal Stem Cells In Mice

Background: Mesenchymal stem cells(MSCs) are characterized by high self-renewal ability, multi-lineage differentiation potentials. Hair follicle derived mesenchymal stem cells(h HF-MSCs) are easily accessible, rich source of autologous tem cells, exhibiting immune privilege and non tumor formation after transplantation. Accumulating research shows h HF-MSCs have been used as cell sources in skin repair and regeneration, engineering of functional small diameter vascular grafts and delivering of human insulin gene in huan gene therapy. It is well-known that the efficacy and safety of stem cell in regenerative medicine largely depend on migration and homing of the cells into damaged sites. However the migration and homing of h HF-MSCs in animal models have not been studied. Aims: The aim of this research is to study the migration and homing of h HF-MSCs in mice. To this end, h HF-MSCs were isolated from human hair follicles and labeled with firefly luciferase and enhanced green fluorescent protein(LG-h HF-MSCs). LG-h HF-MSCs were administrated through NOD/SCID mice tail injection. molecular image, PCR, fluorescent microscopy, flow cytometry and biochemistry assay were performed to inspect the migration and homing of h HF-MSCs in the mice. Methods: 1. Isolation, cultivation and identification of h HF-MSCsHair follicles were pulled out from a scalp of the fetus, cultured in DMEFm-F12 medium containing 10% FBS. When the cells migrated from the hair follicles, those cells with spindle shape in morphology were collected and divided into two parts. One parts of the cells were immune stained with antibodies against CD Markers and subjected to flow cytometry analysis for inspection of surface marker expressions. Another part of the cells were cultured in adipogenic and osteogenic condition media and subjected to Oil Red O and Alizarin Red staining for multipotency assay towards adipocytes and osteoblasts. Those cells with spindle shape that express the surface marker of MSCs and display adipogenic and osteogenic potentials were designated as hair follicle-derived mesenchymal stem cells(h HF-MSCs). 2. Preparation of Luc-e GFP labeled h HF-MSCsLentiviral vector containing two reporter genes Luc-e GFP under the control of EF1 a promoter was constructed and co-transfected with p MDL-g/p, p SRV-rev and p MD2.G into Phoenix 293 Tcells.Viral particles were collected and concentrated from cultured supernatants by ultra-centrifugation and subsequently transduction into h HF-MSCs. The percentage of e GFP-positve cells(LG-h HF-MSCs) was detected by flow cytometry. The correlation line between LG-h HF-MSCs and luciferase activity was set up by plotting the activities with the numbers of LG-h HF-MSCs. 3. Implantation of LG-h HF-MSCsLG-h HF-MSCs were injected into NOD/SCID mice via mice tail vein. After implantation of the cells, molecular image was performed to detect the luciferase activity. HE staining and immune fluorescence staining were performed to inspect the histology and e GFP expression in the tissues of liver and lung. PCR was carried out to detect of ALU gene expressions in the tissues of liver and lung. The body weights were monitored daily. Results: 1. Derivation of h HF-MSCs from hair folliclesCells derived from hair follicle exhibited spindle shaped in morphology and expressed CD105, CD44, CD73, and CD90; Under adipogenic and oesteogenic cultivation these hair follicle derived cell displayed differentiation potentials towards adipocytes and osteoblast. As a consequent these hair follice-derived stem cells were designated as mesenchymal stem cells(h HF-MSCs) 2. LG-h HF-MSCs retains CD markers of MSCs and exhibits multipotencyLentiviral vector encoding Luc-e GFP under the control of EF1 a promoter was constructed and transduced into h HF-MSCs. After transduction, the h HF-MSCs display spindle shape in morphology and retained the expression of surface markers of CD44, CD73, CD90 and CD105 and multipotency towards adipocytes and osteoblasts. In addition bioluminescence imaging(BLI)assay showed a linear relationship between the numbers of LG-h HF-MSCs cell and luciferase signal(R2=0.99). 3. Implantation of LG-h HF-MSCsLG-h HF-MSCs displayed the highest BLI signal in the lung of NOD/SCID mice at days 1, 3, 7 days post-transplantation and the body weights were no significant differences comparable to control group. As expected the intensity of BLI signal in the lung decreased with time, PCR showed ALU gene exclusively expressed in the lung of mice implanted with LG-h HF-MSCs. In consitant with molecular image and PCR assay, immune fluorescence staining showed e GFP and human CD44 expressed in the lung. HE staining and biochemical functional analysis showed mice transplanted with LG-h HF-MSCs displayed normal histological structure in the lung and liver and normal biochemical functionality in the liver, kidney and heart comparable to control group. Conclusions: NOD/SCID mice implanted with h HF-MSCs retained histological structure and biochemical functionality, and h HF-MSCs largely distributed in lung comparable to control group.