Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells

Abstract

Background: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs.

Methodology/principal findings: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments.

Conclusions/significances: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

Figure 1. Establishment of EMSCs and BMMSCs.

(A) Schematic protocol for the establishment of cultures of EMSCs and BMMSCs. (B) Phase-contrast micrograph of EMSCs in primary culture after seven days. Arrow heads indicate triangle, spindle-shaped, fibroblast-like EMSCs. Lymphohematopoietic cells are indicated by arrows. (C) Phase-contrast micrograph of BMMSCs in primary culture after seven days. Arrow heads indicate flat, fibroblast-like BMMSCs and arrows indicate lymphohematopoietic cells. (D) Phase contrast micrograph of EMSCs at the fifth passage showing that EMSCs maintained their shape during propagating. (E) Phase contrast micrograph of BMMSCs upon the fifth passage showing that BMMSCs became flatted with increasing passages. All the scale bars represent 100 µm.

Figure 2. Immunophenotypes of EMSCs and BMMSCs.

(A) Flow cytometric analysis of markers related to stem cells, mesenchymal stem cells, hematopoietic cells, endothelial cells and immune cells on EMSCs and (B) BMMSCs at the first passage. The dotted line indicates the respective isotype control.

Figure 3. Surface markers analysis of EMSCs after IFN-γ treatment and during increased serial passages.

(A) MHC I and MHC II expression profiles for EMSCs (passage five) were analysed under 10 ng or (B) 100 ng IFN-γ treatment. EMSCs were positive for MHC I and MHC II after IFN-γ treatment. (C) EMSCs surface antigen profiles were detected via flow cytometric analysis during increasing passages. The markers of CD73, CD166, SSEA-4 and Sca-1 were decreased with propagating. Respective isotype control is indicated by the dotted line.

Figure 4. Cell proliferation profiles of EMSCs and BMMSCs.

(A) Colony-forming activity of EMSCs and BMMSCs at the first passage. EMSCs formed 23.17±2.92 CFUs whereas BMMSCs formed 5.83±1.60 CFUs (n = 6). (B) CFUs size distribution for EMSCs and BMMSCs. EMSCs formed more and larger CFUs than BMMSCs. ** represents P<0.01 (n = 6). (C) PDTs for EMSCs were compared to BMMSCs at the third (P3) and the fifth (P5) passages. The curves of EMSCs are higher than the ones of BMMSCs (n = 3). * represents P<0.05, ** represents P<0.01, which was compared at the third passage. # represents P<0.05, ## represents P<0.01, which was compared at the fifth passage. (D) Propagating abilities of EMSCs and BMMSCs. Cells were propagated for 2 days at each passage and cell numbers were calculated. EMSCs could be propagated for more passages that cells still retained proliferation ability than BMMSCs (n = 5). * represents P<0.05, ** represents P<0.01, as compared to previous passage. (E) Telomere length and (F) telomerase activity of EMSCs and BMMSCs (at the first passage) were detected and compared. EMSCs expressed significantly longer telomere length and higher telomerase activity than BMMSCs (n = 3). * represents P<0.05, ** represents P<0.01. Data are presented as mean ± s.d. and analyzed with Student's t-test.

Figure 5. Differentiation potential of EMSCs and BMMSCs.

(A) Adipogenic differentiation of EMSCs and BMMSCs at the fifth passage. Adipogenic capability was characterized by Oil Red O staining after seven days induction. EMSCs showed larger and more lipid drops than BMMSCs. (B) Oil Red O staining of EMSCs and BMMSCs were quantified for comparison (n = 3). (C, D) Expression level of adipogenic related genes were assessed by qPCR. EMSCs showed higher adipogenic gene expression level after seven days induction (n = 3). (E) MHC I expression profile of EMSCs was analysed by flow cytometry after seven days adipogenic induction. After adipogenesis, EMSCs were still negative for MHC I expression. (F) Osteogenic differentiation of EMSCs and BMMSCs at the fifth passage. Osteogenic capability was characterized by ARS staining after seven days induction. EMSCs showed higher calcium deposition than BMMSCs. (G) Quantitative results of ARS were performed to compare the osteogenic capacity of EMSCs and BMMSCs (n = 3). (H, I) Expression level of osteogenic related genes were assessed by qPCR. EMSCs showed higher osteogenic gene expression level after seven days induction (n = 3). (J) MHC I expression profile of EMSCs was analysed by flow cytometry after seven days osteogenic induction. After osteogenesis, EMSCs were still negative for MHC I expression. (K) Chondrogenic differentiation of EMSCs and BMMSCs at the fifth passage. Chondrogenic capability was evaluated by histological section of micromass pellet cultures after 21 days induction. Glycosaminoglycan content of the pellet was characterized by toluidine blue staining. EMSCs showed deeper purple stained matrix than BMMSCs. All the scale bars represent 100 µm. ** represents P<0.01. Data are presented as mean ± s.d. and analyzed with Student's t-test.

Figure 6. Analysis of cellular aging related markers of EMSCs and BMMSCs.

(A) Determination of senescence marker, SA-β-gal, upon the fifth passage of EMSCs and (B) BMMSCs. SA-β-gal positive cells were showed as blue color and indicated by arrow heads. (C) The number of SA-β-gal positive cells was calculated from EMSCs or BMMSCs cultures. EMSCs showed lower SA-β-gal positive cells than BMMSCs (n = 4). (D) mRNA levels of cyclin-dependent kinase inhibitors p16 and (E) p21 were evaluated by qPCR. Both p16 and p21 expression levels were lower in EMSCs than BMMSCs (n = 3). All the scale bars represent 100 µm. ** represents P<0.01. Data are presented as mean ± s.d. and analyzed with Student's t-test.

Figure 7. Immunomodulatory properties of EMSCs.

(A) Responding splenocytes from C57BL/6 or (B) BALB/c mice were stimulated with Con A in the presence or absence of graded numbers of C57BL/6 EMSCs. The results are shown as percentage of cell proliferation in comparison with control cell proliferation. Splenocytes proliferation was inhibited by culture with EMSCs (at the fifth passage) in a dose dependent manner (n = 3). (C) Responding splenocytes from C57BL/6 mice were cultured with an equal number of mitomycin C-treated BALB/c splenocytes and with or without graded number of C57BL/6 EMSCs (n = 3). Allo indicates allogeneic splenocytes culture. EMSCs inhibited the splenocyte proliferation stimulated by allogeneic cells in a dose dependent manner. (D–F) Analysis of inflammatory cytokine production of splenocyte stimulated by Con A. Inflammatory cytokines expression level of splenocytes were decreased when co-cultured with EMSCs (at the fifth passage) (n = 3). * represents P<0.05, **represents P<0.01. Data are presented as mean ± s.d. and analyzed with Student's t-test.

Figure 8. Repair of bone fracture in mice after transplantation of EMSCs.

(A) Fracture healing was assessed by X-ray after 14 days with or without EMSCs (at the fifth passage) transplantation. Arrowheads indicate the site of fracture. (B) The bone density of fracture site (dotted box in Figure 8A) was quantified (n = 4). EMSCs transplantation significantly improved osteocalcification as compared to the control group. (C) The morphology of fracture sites were examined by H&E staining. Tissue disorganization was showed in the control group while the EMSCs transplanted group newly formed bone tissue (arrow) and capillaries (arrowhead). * represents P<0.05. Scale bars represent 100 µm. Data are presented as mean ± s.d. and analyzed with Student's t-test. Control: SPONGOSTAN carried with complete medium-transplantation group; EMSC: SPONGOSTAN carried with EMSCs-transplantation group.

Figure 9. Prevention of skin flap necrosis in mice by EMSCs injection.

(A) Ischemic flap showed necrosis in the control mice and near complete healing in the EMSCs (at the fifth passage) injected mice after 6 days of surgery. (B) Percentage of necrosis was quantified by planimetry. Percentage of necrotic area is significantly larger in the control group than in the EMSCs injected group (n = 4). (C) The skin structure was evaluated by H&E staining. Skin thickness is decreased and mucinous layer is preserved in the EMSCs injected group as compared to the control group. (D) In higher magnification, extra vascular erythrocytes and hemorrhage (arrowhead) can be observed in the control group. (E) Immunostaining of von Willebrand factor (vWF) of skin flap, and (F) its quantification showed significantly more vWF expressed cells (arrowhead) in the EMSCs injected group than the control group. **represents P<0.01. Scale bars represent 100 µm. Data are presented as mean ± s.d. and analyzed with Student's t-test. PM: platysma muscle; ML: mucinous layer; Control: complete medium injection-group; EMSC: EMSCs-injection group.

Figure 10. Transplanted EMSCs improved blood perfusion in ischemic limb.

(A) Foot perfusion was evaluated by laser Doppler blood perfusion analysis at day 7 post ischemia. In color-coded images, red represents normal perfusion, while dark blue represents low or absent perfusion. (B) Quantification of the foot perfusion showed that EMSCs (at the fifth passage) transplantation significantly improved blood perfusion (n = 3). (C) H&E staining showed massive muscle degeneration in ischemic regions in the control group compared to the markedly reduced muscle degeneration in the EMSCs group. (D) Masson's Trichrome staining and (E) its quantification showed significantly larger fibrotic area in the control group than the EMSCs injected group. (F) Immunostaining of von Willebrand factor (vWF) of ischemic limb muscle and (G) its quantification showed significantly more vWF expressed cells (arrowhead) in the EMSCs injected group than the control group. Scale bars represent 100 µm. **represents P<0.01. Data are presented as mean ± s.d. and analyzed with Student's t-test. Isch: ischemic limb; NI: non-ischemic limb; Control: complete medium injection-group; EMSC: EMSCs-injection group.