Islation,in Vitro Culture And Identification Of Bone Marrow Derived Renal Stem Cells In Ishemically Injured Kidney

Objective:By establishing renal ischemia-reperfusion (I/R) injury model in murine undergoing bone marrow transplantation (BMT), we investigated whether bone marrow (BM) derived stem cells could convert into renal stem cells following their engraftment to damaged kidney. We also examined the beneficial effects on this conversion induced by granulocyte-colony stimulating factor (G-CSF). We isolated bone marrow derived renal stem cells (BMRSCs) from damaged kidney and evaluated their phenotype and expansive potential in in vitro culture. Our study aims to provide novel theoretical evidence for contribution of bone marrow derived cells in renal repair.Methods:Bone marrow cells isolated from the transgenic C57BL/6J mice labeled with green fluorescent protein (GFP) were injected into normal C57BL/6J recipient mice which in advance undergo lethal137Cs irradiation. After the confirmation of hematopoietic reconstruction, we employed unilateral renal I/R injury in recipient mice. Subsequently, we examined the migration of bone marrow derived cells/stem cells into damaged kidney in different time point following I/R injury by flow cytometry. Importantly, we detected the dynamic percentage of GFP+CD45+cells in kidney to provide the evidence of the conversion of BM derived stem cells to renal stem cells. To investigate whether G-CSF mobilization could enhance the recruitment of BM derived cells/stem cells into injured kidney, the recipient mice were divided into two groups:G-CSF-and saline-treated group. Furthermore, we harvested the damaged kidney2months after I/R injury. Renal cells were obtained by digestion and subjected to in vitro culture. We tested the proliferative potential of GFP+BMRSCs and assessed their phenotypic properties. Moreover, the effect of fibronectin on the proliferative capacity and plastic adherent ability of BMRSCs in culturing system was also evaluated.Results:Four weeks after BMT, the percentage of GFP+cells among peripheral blood mononuclear cells in recipient mice was detected as81.83%-97.31%(90.93±3.19%). After G-CSF mobilization, the ratio of the expression of several stem cell markers (CD90, CD133, FLK-1) on peripheral blood mononuclear cells in G-CSF-administered group were11.38±0.81%,8.95±0.80%and2.20±0.29%, respectively, which apparently higher than those in saline-treated group (2.11±0.60%,2.12±0.66%and1.38±0.30%). The difference is statistically significant. Subsequently, our results revealed that the engrafted BM derived (GFP+) cells in kidney increased gradually post I/R injury. At an early time point (14days after injury), the percentages of GFP+cells in saline-treated contralateral kidney (SCK), saline-treated injured kidney (SIK), G-CSF-treated contralateral kidney (GCK), and G-CSF-treated injured kidney (GIK) were2.75±0.55%,5.77±0.33%,2.40±0.35%, and4.72±0.46%, respectively, showing that SIK>SCK, GIK>GCK (with statistical difference) and SIK>GIK (with no statistical difference). At a late time point (28days after injury), the percentages of GFP+cells in SCK, SIK, GCK, and GIK were3.35±0.35%,9.43±0.43%,6.15±0.37%, and15.98±1.22%, respectively, displaying that SIK>SCK, GIK>GCK and GIK>SIK (all with statistical difference). The migration of GFP+cells in kidney at56-day time point is similar to that of28days post injury. Moreover, our study demonstrated an increasing number of BM derived stem cells (GFP+Sca-1+) after I/R injury. At an early time point, the percentages of GFP+Sca-1+cells in ischemic kidneys were higher than that in contralateral kidneys in both G-CSF-and saline-treated groups, although the difference was not statistically significant. Twenty-eight days after injury, the percentages of GFP+Sca-1+cells in SCK, SIK, GCK, and GIK were1.75±0.30%,5.15±0.75%,2.60±0.60%, and9.55±1.32%, respectively, disclosing that SIK>SCK, GIK>GCK, and GIK>SIK (all with statistical difference). The migration of GFP+Sca-1+cells in kidneys at56-day time point appears similar to that of28days post injury. The expression of GFP+CD133+and GFP+CD44+on intrarenal cells resembled that of GFP+Sca-1+cells. Two weeks post renal injury, the percentages of GFP+CD45+cells in ischemic kidneys were higher than that in contralateral kidneys in both groups with no statistically significant difference. Four weeks after injury, the percentages of GFP+CD45+cells in SCK, SIK, GCK, and GIK were2.25±0.35%,7.50±0.31%,4.42±0.43%, and13.00±1.03%, respectively, revealing that SIK>SCK, GIK>GCK, and GIK>SIK (all with statistical difference). However, eight weeks after injury, the percentages of GFP+CD45+cells in SCK, SIK, GCK, and GIK were4.72±0.69%,2.20±0.39%, 3.00±0.54%, and1.70±0.26%, respectively, exhibiting that SCK>SIK, GCK>GIK (with statistical difference), and SIK>GIK (with no statistical difference). Comparing to the percentage of GFP+CD45+cells in kidneys at4-week time point, this population of cells decreased apparently in GIK, SIK (with statistical difference) and GCK (with no statistical difference), whereas increased in SCK (with no statistical difference) at8-week time point.In in vitro culture, BMRSCs were round or oval in morphology and demonstrated a clonogenicity as well as a gathering growth pattern, with an expansive potential for more than30days. The percentages of GFP+/GFP+Sca-1+cells among the kidney derived cells increased gradually during the subculture (passage0-6). In in vitro culture, the expression of CD45in BMRSCs was down regulated than that in the newly isolated BMRSCs. Moreover, no expression of CD34or CD90was seen in BMRSCs. Comparing to the culture system without Fn (8.33±0.81dys), the average days consumed per passage was significantly reduced in Fn coated culturing (5.40±0.46dys), with a statistical difference. Furthermore, the percentage of GFP+cells (constituently BMRSCs) was apparently higher in Fn coated culturing (1.96±0.24%) than in culture system without Fn (0.92±0.17%), with a statistical difference.Conclusions:1) G-CSF is capable of mobilizing BM derived stem cells into peripheral blood and enhance the homing of BM derived cells/stem cells to the damaged kidney.2) BM derived stem cells undergoes a conversion into BMRSCs at late time point post renal injury, which can be enhanced by G-CSF.3) In in vitro culture, BMRSCs demonstrate a higher proliferative potential than resident renal stem/progenitor cells.4) In in vitro culture, BMRSCs lose the markers of hematopoietic cells/stem cells, while retaining the expression of Sca-1.5) In in vitro culturing system, the use of Fn facilitates the adherence and enhances the proliferative potential of BMRSCs.