Caused By Total Body Irradiation In Mice Of Flk-1 + Bone Marrow Mesenchymal Stem Cells Aging Related Osteogenic Potential Changes And Their Relationship With The Bones And Hematopoietic System

Stem cells are located throughout the embryonic and adult body of higherorganisms with unique abilities of self-renewal and multipotentiai differentiation.The growing body of information have evidenced the significant role of adultstem cells (ASCs), especially mesenchymal stem cells (MSCs), in supporting acontinuous renewal and regeneration of tissues in homeostasis and in response todisease or injury.We isolated and identified previously Flk-1⁺MSCs in diverse human fetaltissues and organs (bone marrow (BM), heart, liver, lung, kidney, muscle, dermaand pancreas) as well as in murine BM. They had similar morphology andphenotype (positive for Flk1, CD29, CD44, CD105 and lack the expression ofendothelial and hematopoietic antigens, such as vWF,CD31,CD34,CD11a,CD11b and HLA-DR) and were able to differentiate intocells with morphologic and phenotypic characteristics of adipocytes, osteoblasts,neurons, glia and hepatocytes. Furthermore, the role they played in tissueregeneration had been evidenced in several disease/injury models. Moreimportantly, we found that Flk-1⁺MSCs also existed in BM of patients withchronic myeloid leukemia (CML), aplastic anemia (AA), multiple myeloma (MM)and meylodysplastic syndrom (MDS). Although with similar morphologic andphenotypic characteristics, these Flk-1⁺MSCs in CML carry the BCR/ABLfusion gene or have abnormal differentiation and immunoregnlatory potential inother diseases mentioned above, which all contribute to the occurrence andprogression of these diseases. Therefore, Flk-1⁺MSCs participate not only intissue regeneration and repairing but also in response to disease or injury, theabnormal biological characteristics of Flk-1⁺ MSCs may also be the pathogenesisat stem cell lever of some diseseas.Ionizing irradiation (IR) is often used in treatment for cancer patients.Unfortunately, this therapy is not tumor-specific. Normal tissues, particularly the bone and hematopoietic system are vulnerable to cytotoxicity caused by IR,resulting in some short- and long-term complications. Reports have shown thatIR could induce cellar senescence in hematopoietic stem cells (HSCs) which maybe the underlying mechanisms of long-term hematopoietic injury. Apart fromHSCs, MSCs also reside in the bone cavity, proposed to give rise to the majorityof HSCs microenvironment cell lineages which constitute the HSCs niche.Furthermore, osteoblasts, originating from BMMSCs, not only play importantrole in the bone remolding but also are the key cellular component of HSCs niche.Therefore, a murine total body irradiation (TBI) model was applied in thefollowing experiment to examine the effect of TBI on Flk1⁺BMMSCs and therelated clinical significance.The purpose of the first part of this paper is to investigate whether TBI couldinduce cellular senescence in Flk1⁺BMMSCs. Our results showed that after asingle 4Gy TBI no cellular senescence related morphology and increase insenescence-associatedβ-galactosidase (SA-β-gal) were found. Contrast to cellcycle arrest in senescent cells, more Flk1⁺BMMSCs entered S phase of cell cycleat day 3 after TBI, followed by recovery of cell cycle distribution to normal level.Furthermore, TBI could not increase either the expression of senescence-relatedgenes such as p16^INK4a, p21^Cip1/waf1, p53 and transforming growthingfactor-β1(TGF-β1).Owing to the pivotal roles osteoblasts played in bone and HSCs niche, thesecond part of our research focuses on the effect of TBI on the BM mesenchymalstem/progenitor cells pool and their osteogenic potential. We found that 4Gy TBIreduced the BM mesenchymal stem/progenitor cells pool significantly asmanifested by the reduction of CFU-F after TBI. Meanwhile, TBI altered theosteoblast differentiation ability of Flk1⁺BMMSCs, that was shortly afterirradiation (day 1 and 3 after TBI), the osteogeneic differentiation potentialincreased significantly, with more ALP-positive cells and a calcium-richermineralized matrix in the induction dish, which also was evidenced by the changes in the expression of osteogenesis related genes detected by real-timeRT-PCR. The changes in TAZ (transcriptional coactivator with PDZ-bindingmotif) expression in Flk1⁺BMMSCs may be contribute partially to the alteredosteogenic potential.In the third part, the changes in the number of osteoblasts, bone and HSCsniche in vivo were examined to explore the influence of damage of quantity andquality of Flk1⁺BMMSCs on bone and hematopoietic system. It was obvious thatafter 4Gy TBI, the number of osteoblasts decreased, accompanied with thedisruption of the bone histomorphometric and reduction in bone mineral density(BMD). Furthermore, the osteoblastic niche pool also reduced greatly, reflectedby the reduction of the functional cellular component: spindle-shapedN-cadherin-expressing osteoblasts (SNOs).In conclusion, our study has shown that TBI induced significant shrinking ofBM mesenchymal stem/progenitor cells pool as well as obvious alteration inosteogeneic potential which occur by a senescence-independent mechanism. Theenhanced osteogeneic potential and entery of more Flk1⁺BMMSCs into S phaseof cell cycle at early phase indicated that Flk1⁺BMMSCs participate in BMmaintenance or regeneration in response to irradiation stress. But thiscompensation is limited and the following reduction in quantity and osteogeneicpotential of Flk1⁺BMMSCs might be the underlying mechanisms at the stem celllevel that primarily are responsible for the long-term bone loss and also partiallycontribute to haematopoietic injury after TBI.