Mesenchymal stem cell-mediated ectopic hematopoiesis alleviates aging-related phenotype in immunocompromised mice

Abstract

Subcutaneous transplants of bone marrow mesenchymal stem cells (BMMSCs) are capable of generating ectopic bone and organizing functional hematopoietic marrow elements in animal models. Here we report that immunocompromised mice received subcutaneous BMMSC transplants using hydroxyapatite tricalcium phosphate as a carrier suppressed age-related degeneration in multiple organs and benefited an increase in life span extension compared with control littermates. The newly organized ectopic bone/marrow system restores active hematopoiesis via the erythropoietin receptor/signal transducer and activator of transcription 5 (Stat5) pathway. Furthermore, the BMMSC recipient mice showed elevated level of Klotho and suppression of insulin-like growth factor I signaling, which may be the mechanism contributing to the alleviation of aging-like phenotypes and prolongation of life in the treated mice. This work reveals that erythropoietin receptor/Stat5 pathway contributes to BMMSC-organized ectopic hematopoiesis, which may offer a treatment paradigm of reversing age-related degeneration of multiple organs in adult immunocompromised mice.

Figure 1

Subcutaneous transplantation of human BMMSCs extends life span in immunocompromised mice. (A) Kaplan-Meier analysis of survival. Recipient mice transplanted with subcutaneous human BMMSC using HA/TCP as a carrier vehicle (Transplant, n = 13) manifested a significantly increased life span compared with age-matched control immunocompromised mice (Control, n = 20; P < .05). (B) The natural aging phenotype of human kyphosis was observed in 6 of 9 aging control mice, shown here as extreme curvature of the vertebrae and tail, although not in transplantation mice (n = 15). (C) Body weight at indicated months showed no significant difference between control and transplant recipients. Bars represent the means. (D) When human skin fibroblasts were transplanted subcutaneously using HA/TCP as a carrier (HA/TCP/FB), there is no increased life span in the recipients (n = 10; P = .391). (E) Newly formed bone (B) and bone marrow (BM) were found in the BMMSC transplants (Transplant) at 8 weeks after transplantation. However, the fibroblast group (HA/TCP/FB) failed to form new tissue and only showed connective tissue (CT) around HA/TCP particles (HA) by hematoxylin and eosin staining. (F) When human BMMSCs (10⁶) were infused into immunocompromised mice (n = 12) via tail vein, there was no consistent increase in life span extension (P > .05) compared with the control group.

Figure 2

EPO-R is a progenitor marker of human BMMSCs and mediates bone marrow organization in vivo. (A) Flow cytometric analysis revealed that a small percentage (5.54%) of BMMSCs expressed EPO-R. (B) RT-PCR analysis confirmed EPO-R gene expression in BMMSCs. Jurkat cells were used as a positive control for EPO-R expression. (C) Western blot analysis further confirmed that BMMSCs express EPO-R at passages 1, 5, and 10 (P1, P5, and P10). (D) rhEPO treatment (0.1 U/mL) for the indicated time (minutes) induced a significantly up-regulated expression of phospho-Stat5 in BMMSCs at 30 minutes compared with either β-actin or Stat5 (n = 3). However, expression level of Stat5 showed no significant change (n = 3). (E) STRO-1⁺, CD146⁺, and CD166⁺ BMMSCs were significantly increased in the rhEPO treatment group (EPO⁺) compared with the untreated group (EPO⁻; n = 3). (F) rhEPO (0.1 U/mL)–treated BMMSCs (EPO⁺) were capable of inducing active hematopoietic marrow formation (arrows) when transplanted into immunocompromised mice with HA/TCP (HA) as assessed by hematoxylin and eosin staining. B indicates bone. Original magnification ×400. Bars represent SD (EPO⁺, n = 3; EPO⁻, n = 3). Semiquantitative analysis showed that EPO treatment resulted in a significantly increased bone marrow formation in the BMMSC transplants compared with untreated control BMMSCs. (G) Western blotting analysis confirmed a significant inhibition of EPO-R and Stat5 expression in BMMSCs transfected with siRNA targeting EPO-R and Stat5, respectively. (H) Loss of function of EPO-R and Stat5 resulted in inhibition of bone marrow (arrow) formation in 8-week-old BMMSC transplants compared with the nonspecific siRNA-transfected transplant (Control). HA indicates HA/TCP. Original magnification ×200. Bars represent SD (Control, n = 3; EPO-R, n = 3; Stat5, n = 3; ***P < .005 vs Control; #P < .05 vs EPO-R; ###P < .005 vs EPO-R).

Figure 3

Subcutaneous transplantation of human BMMSCs reconstitutes active hematopoiesis in adult immunocompromised mice. (A) Micro-computed tomography analysis revealed that bone (B) and bone marrow elements (BM) regenerated in 8-week BMMSC transplants. Original magnification ×200. (B,C) Long-term (12 months) engraftment of BMMSC-mediated bone/marrow formation in subcutaneous transplants showing organized bone (B) and bone marrow components (BM) by hematoxylin and eosin staining (B) and BrdU label retaining assay showing BrdU-positive cells (arrows) in the bone marrow (BM) compartment for 14 weeks after labeling (C). Original magnification ×200. (D) A scheme of eGFP⁺ mouse bone marrow (BM) cells homed to the BMMSC-generated bone/marrow organs. BMMSCs were transplanted subcutaneously into immunocompromised mice (top panel). Eight weeks after transplantation, eGFP⁺ BM cells were injected through the tail vein of the primary transplant recipient (second top panel). The eGFP⁺ BM cells homed to the bone marrow niche in the primary BMMSC transplants (third top panel). Four weeks later, the primary BMMSC transplants were removed as donor transplants for secondary transplantation (fourth top panel). Four weeks after secondary transplantation, peripheral blood was collected for flow cytometric analysis (bottom panel). (E) The secondary transplantations were capable of supplying hematopoietic cells in the circulation of the recipients (n = 3) at 4 weeks after transplantation. Flow cytometric analysis revealed the existence of eGFP lymphocytes, monocytes, and polymorphonuclear leukocytes in peripheral blood leukocytes (PBLs). PBLs of the nontransplanted mice were used as negative controls. An average of 3 mice per group was used in the analysis of each cell subset. Comparative analysis of each cell subset was determined and statistically analyzed (Control, n = 3; Transplant, n = 3; leukocyte percentage, P < .01; R1, P < .005; R2, P < .05; R3, P < .005). Data are representative of 3 independent experiments.

Figure 4

Subcutaneous transplantation of human BMMSCs rescues bone marrow elements. (A) Representative femur sections showed increment of trabecular bone in 3 BMMSC transplant recipient mice at 16 months of age (Transplant) compared with the untreated age-matched littermates (Control; n = 3). Abundant red marrow elements, characteristic of increased hematopoietic cells, were observed in the transplant recipients compared with the fatty, acellular marrow compartment in the control mice. EP indicates epiphyseal cartilage; TB, trabecular bone. *Bone marrow. Original magnification ×200. (B) Immunohistochemical staining showed numerous CD45⁺, B220⁺, and TER119⁺ cells (open arrows) in the femur bone marrow of recipient mice (Transplant, n = 3) compared with the age-matched control mice (Control, n = 3). Bars represent SD. (C) BMMSCs isolated from mice that received subcutaneous BMMSC transplants (n = 3) were transplanted into new immunocompromised recipients mice using HA/TCP (HA) as a carrier. At 8 weeks after transplantation, the transplants showed significantly increased bone (top panel) and bone marrow (bottom panel) formation compared with the BMMSC transplants from control mice (n = 3). Bars represent SD. (D) Immunohistochemical staining showed increased CD45⁺, B220⁺, and TER119⁺ cells (arrows) in the ectopic bone marrow compartment (BM) generated by BMMSCs from the mice received subcutaneous BMMSC transplants (Transplant; n = 3) compared with the ectopic bone marrow compartment (BM) generated by BMMSCs from regular mice (Control, n = 3). B indicates bone; HA, HA/TCP. Bars represent SD.

Figure 5

Subcutaneous transplantation of human BMMSCs rescues bone loss. (A) Micro-computed tomography analysis revealed that the third lumbar vertebra in transplant recipient mice (Transplant, lower panels) at 10 months after transplantation showed increase in trabecular bone volume compared with that of age-matched control mice (Control, top panels). B indicates vertebral body; Sp, spinosus process of vertebra. *Vertebral foramen. (B) Bone mineral density (BMD) of femurs in recipient mice (Transplant, n = 3) was significantly improved compared with age-matched controls (Control, n = 3), as assessed by dual X-ray absorptiometry analysis. Bars represent SD. (C) Bone morphologic analysis demonstrated increase in bone volume versus total volume (BV/TV) and trabecular number (Tb.N) and decrease in trabecular separation (Tb.Sp) in recipient mice (Transplant, n = 3) compared with the controls (Control, n = 3). Bars represent SD. (D) The number of TRAP-positive cells (arrows) decreased in the vertebral body of recipient mice (Transplant, n = 3) compared with control mice (Control, n = 3). Original magnification ×400. Bars represent SD. (E) ELISA assay revealed that serum sRANKL and C-terminal telopeptides type I collagen were decreased in recipient mice (Transplant, n = 3) compared with control mice (Control, n = 3). However, OPG was markedly increased in the recipient mice. Bars represent SD. (F) The number of CFU-F of BMMSCs derived from the recipient mice (Transplant, n = 3) increased compared with the age-matched control (Control, n = 3). Bars represent SD. (G) The proliferation of recipient BMMSCs (Transplant, n = 3) was significantly increased compared with the control group (n = 3), as determined by BrdU incorporation assay. Bars represent SD. (H) The population doublings of BMMSCs from recipient mice (Transplant, n = 3) were significantly increased compared with control mice (Control, n = 3). Bars represent SD. (I) Alizarin red staining showed that BMMSCs derived from recipient mice (Transplant, n = 3) had higher calcium accumulation than that from control mice (Control, n = 3) under osteogenic conditions. Bars represent SD.

Figure 6

Subcutaneous human BMMSC transplantation up-regulated Klotho expression in immunocompromised mice. (A) Immunohistochemical staining with anti-Klotho antibody showed that transplant recipient mice (Transplant) expressed higher level of Klotho () in the epithelial cells of the renal tubules (RT) than those of age-matched controls (Control). G indicates glomerulus. Original magnification ×200. Bars represent SD. (B) Western blot analysis confirmed that Klotho was elevated in kidney of transplant recipient (Transplant, n = 3) compared with nontreated littermates (Control, n = 3). (C) Likewise, Western blot analysis showed elevated Klotho in brain of transplant recipient mice (Transplant n = 3) compared with the Control (n = 3). (D) ELISA assay further confirms elevated serum Klotho level in transplant recipient mice (Transplant, n = 3) compared with the control group (Control, n = 3). Bars represent SD. (E) Random blood glucose measurement showed decreased serum glucose in recipient mice (Transplant, n = 3) in comparison to control mice (Control, n = 3). Bars represent SD. (F) Similarly, serum IGF-I level was lower in recipient mice (Transplant, n = 3) compared with control mice (Control, n = 3). Bars represent SD. (G) Urine protein level was significantly decreased in recipient mice (Transplant, n = 3) compared with control mice (Control, n = 3). Bars represent SD. (H) Western blot analysis showed that expression of insulin receptor (IR) α, IRβ, phosphatidylinositol 3-kinase (PI3-K) p85, and PI3-K p110 was down-regulated in kidney tissues of transplant recipient mice (Transplant, n = 3) compared with control mice (Control, n = 3). β-Actin was used as protein loading control.