Xiao-Yan Wang

A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist in the marrow cavities and the inner interfaces of the bones. The procedure includes flushing bone marrow out of the long bones, digesting the bone chips with collagenase type II, deprivation of the released cells and culturing the digested bone fragments, out of which fibroblast-like cells migrate and grow in the defined medium. The entire technique requires 5 d before the adherent cells are readily passaged. Further identification assays confirm that these cells are MSCs. We provide an easy and reproducible method to harvest mouse MSCs that does not require depletion of hematopoietic cells by sorting or immunomagnetic techniques.

CCR7 guides migration of mesenchymal stem cell to secondary lymphoid organs: a novel approach to separate GvHD from GvL effect.

Inefficient homing of systemically infused mesenchymal stem cells (MSCs) limits the efficacy of existing MSC‐based clinical graft‐versus‐host disease (GvHD) therapies. Secondary lymphoid organs (SLOs) are the major niches for generating immune responses or tolerance. MSCs home to a wide range of organs, but rarely to SLOs after intravenous infusion. Thus, we hypothesized that targeted migration of MSCs into SLOs may significantly improve their immunomodulatory effect. Here, chemokine receptor 7 (CCR7) gene, encoding a receptor that specifically guides migration of immune cells into SLOs, was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (MSCs/CCR7). We found that infusion of MSCs/CCR7 potently prolonged the survival of GvHD mouse model. The infused MSCs/CCR7 migrate to SLOs, relocate in proximity with T lymphocytes, therefore, potently inhibited their proliferation, activation, and cytotoxicity. Natural killer (NK) cells contribute to the early control of leukemia relapse. Although MSCs/CCR7 inhibited NK cell activity in vitro coculture, they did not impact on the proportion and cytotoxic capacities of NK cells in the peripheral blood of GvHD mice. In an EL4 leukemia cell loaded GvHD model, MSCs/CCR7 infusion preserved the graft‐versus‐leukemia (GvL) effect. In conclusion, this study demonstrates that CCR7 guides migration of MSCs to SLOs and thus highly intensify their in vivo immunomodulatory effect while preserving the GvL activity. This exciting therapeutic strategy may improve the clinical efficacy of MSC based therapy for immune diseases. Stem Cells 2014;32:1890–1903

Identification of High Proliferative Potential Precursors with Hemangioblastic Activity in the Mouse Aorta‐Gonad‐ Mesonephros Region

Hemangioblast, a precursor possessing hematopoietic and endothelial potential, is identified as the blast colony‐forming cell in the murine gastrulating embryos (E7.0–E7.5). Whether hemangioblast exists in the somite‐stage embryos is unknown, even though hemogenic endothelium is regarded as the precursor of definitive hematopoiesis in the aorta‐gonad‐mesonephros (AGM) region. To address the issue, we developed a unique three‐step assay of high proliferative potential (HPP) precursors. The AGM region contained a kind of HPP precursor that displayed hematopoietic self‐renewal capacity and was able to differentiate into functional endothelial cells in vitro (i.e., incorporating DiI‐acetylated low‐density lipoprotein, expressing von Willebrand factors, and forming network structures in Matrigel). The clonal nature was verified by cell mixing assay. However, the bilineage precursor with high proliferative potential—the HPP‐hemangioblast (HA)—was not readily detected in the yolk sac (E8.25–E12.5), embryonic circulation (E10.5), placenta (E10.5–E11.5), fetal liver (E11.5–E12.5), and even umbilical artery (E11.5), reflective of its strictly spatial‐regulated ontogeny. Expression of CD45, a panhematopoietic marker, distinguished hematopoietic‐restricted HPP–colony‐forming cell from the bipotential HPP‐HA. Finally, we revealed that basic fibroblast growth factor, other than vascular endothelial growth factor or transforming growth factor‐β1, was a positive modulator of the HPP‐HA proliferation. Taken together, the HPP‐HA represents a novel model for definitive hemangioblast in the mouse AGM region and will shed light on molecular mechanisms underlying the hemangioblast development.

Tumor Necrosis Factor-α Alters the Modulatory Effects of Mesenchymal Stem Cells on Osteoclast Formation and Function

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11b(+) monocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor-alpha (TNF-alpha)-stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-alpha-stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-alpha-stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF-alpha levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases.

Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region

Background The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0–7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined. Design and Methods The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity. Results Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19+ B lymphocytes, and CD3+TCRβ+ T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31+ tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31+, endomucin+ and CD105+ subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis. Conclusions The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.

Deubiquitinase MYSM1 Is Essential for Normal Bone Formation and Mesenchymal Stem Cell Differentiation

Deubiquitinase MYSM1 has been shown to play a critical role in hematopoietic cell differentiation and hematopoietic stem cell (HSC) maintenance. Mesenchymal stem cells (MSCs) are multipotent stromal cells within the bone marrow. MSCs are progenitors to osteoblasts, chondrocytes, adipocytes, and myocytes. Although, MSCs have been extensively studied, the roles of MYSM1 in these cells remain unclear. Here we describe the function of MYSM1 on MSC maintenance and differentiation. In this report, we found that Mysm1−/− mice had a lower bone mass both in long bone and calvaria compared with their control counterpart. Preosteoblasts from Mysm1−/− mice did not show changes in proliferation or osteogenesis when compared to WT mice. Conversely, Mysm1−/− MSCs showed enhanced autonomous differentiation and accelerated adipogenesis. Our results demonstrate that MYSM1 plays a critical role in MSC maintenance and differentiation. This study also underscores the biological significance of deubiquitinase activity in MSC function. Mysm1 may represent a potential therapeutic target for controlling MSC lineage differentiation, and possibly for the treatment of metabolic bone diseases such as osteoporosis.

SOCS1 Regulates the Immune Modulatory Properties of Mesenchymal Stem Cells by Inhibiting Nitric Oxide Production

Mesenchymal stem cells (MSCs) have been shown to be highly immunosuppressive and have been employed to treat various immune disorders. However, the mechanisms underlying the immunosuppressive capacity of MSCs are not fully understood. We found the suppressor of cytokine signaling 1 (SOCS1) was induced in MSCs treated with inflammatory cytokines. Knockdown of SOCS1 did not bring much difference on the proliferation and differentiation properties of MSCs. However, MSCs with SOCS1 knockdown exhibited enhanced immunosuppressive capacity, showing as inhibiting T cell proliferation at extremely low ratio (MSC to T) in vitro, significantly promoting tumor growth and inhibiting delayed-type hypersensitivity response in vivo. We further demonstrated that SOCS1 inhibited the immunosuppressive capacity of MSCs by reducing inducible nitric oxide synthase (iNOS) expression. Additionally, we found the significantly lower SOCS1 expression and higher nitric oxide (NO) production in MSCs isolated from synovial fluid of rheumatoid arthritis patients. Collectively, our data revealed a novel role of SOCS1 in regulating the immune modulatory activities of MSCs.

Migration of dorsal aorta mesenchymal stem cells induced by mouse embryonic circulation

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid‐gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45−CD31−Flk‐1−CD44+CD29+), expressed perivascular markers (α‐SMA+NG2+PDGFRβ+PDGFRα+), and had tri‐lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5–E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA‐MSCs through platelet‐derived growth factor (PDGF) receptor‐, transforming growth factor‐beta receptor‐, but not basic fibroblast growth factor receptor‐mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood‐ or PDGF‐mediated migration of DA‐derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis. Developmental Dynamics, 2011.

Co-culture of Spleen Stromal Cells with Bone Marrow Mononuclear Cells Leads to the Generation of A Novel Macrophage Subset

Macrophages adopt diverse activation states depending on the microenvironment. Recently, stromal cells have been demonstrated to be organizers of the microenvironment. Here, using splenic stromal cells to mimic the splenic microenvironment in vivo, we show that spleen stromal cells can programme bone marrow‐derived mononuclear cells to differentiate and polarize into a novel macrophage subset. These differentiated macrophages (Diff‐Mφ) exhibited pronounced production of IL‐10, IL‐6 and TNF‐α, but diminished the production of IL‐12 in response to LPS. The generation of Diff‐Mφ depended on cell–cell contact as well as on soluble factors. Diff‐Mφ directly suppressed the antigen‐non‐specific (CD3/CD28) CD4+ T cell proliferative response and induced cell death of activated CD4+ T cells. As for cytokine production in CD4+ T cells, Diff‐Mφ promoted IL‐10 and IL‐17 production, whereas inhibited IL‐4 production and did not alter IFN‐γ production. Besides, Diff‐Mφ also expressed iNOS, CD16/CD32, CD54, CD43, CCR7, CD44, PD‐L1 and FasL, which might be involved in the function of Diff‐Mφ. These results suggest that splenic microenvironment may physiologically induce a novel type of macrophages differentiation.

Stem cell hierarchy in dorsal aorta

During embryogenesis, the aorta-gonad-mesonephros (AGM) region serves as an important niche for various kinds of stem cells, including hematopoietic stem cells (HSCs), primordial germ cells (PGCs), meso-angioblasts and angioblasts. Here, we report additional two types of stem cells that are in close association with definitive hematopoiesis in the AGM. While the hemogenic endothelium is well known as the precursor of AGM hematopoiesis, the hemangioblast, a cell type bearing hematopoietic and endothelial potential, is hypothesized for long time. To address the issue, we developed a unique three-step assay of high proliferative potential (HPP) precursors. The dorsal aorta plus its surrounding mesenchyme (AoM) other than the gonad and mesonephros (GM) contained clonal HPP precursors (CD45+ CD31–) with hemangioblastic characteristics. They displayed hematopoietic self-renewal ability and were readily to differentiate into functional endothelial cells in vitro, i.e. incorporating Ac-LDL and forming network structures in Matrigel. The clonal nature was verified by cell mixing assay. Surprisingly, the bipotential precursor, designated here as the HPP-HA, was not readily detected in the yolk sac (E8.25-E12.5), embryonic circulation (E10.5), placenta (E10.5-E11.5), fetal liver (E11.5-E12.5) and even umbilical artery (E11.5), reflective of its strictly spatial-regulated ontogeny. Intriguingly, the HPP-HA was detected in the umbilical artery when cocultured with AGM cells, suggesting the unique microenvironment cues in the AGM. Mesenchymal stem cells (MSCs) are multi-potent stem cells that can generate various microenvironment components in bone marrow, ensuring a tight and precise control over self-renewal and multi-lineage differentiation of HSCs. Nevertheless, their spatiotemporal correlation with embryonic hematopoiesis remains unclear in mammalian embryos. We find that human AoM resided with bona fide MSCs. They were highly self-renewal as fibroblastoid population bearing uniform surface markers (CD45–, CD34–, SH2+, SH3+, CD29+, and CD44+), expressed pluri-potential molecules Oct-4 and Nanog, and clonally demonstrated tri-lineage differentiation capacity (osteocytes, chondrocytes and adipocytes). Moreover, they were functionally equivalent to MSCs from adult bone marrow, i.e., maintaining long-term hematopoiesis and suppressing T lymphocyte proliferation in vitro. In comparison, the yolk sac contained bi-potent mesenchymal precursors that propagated more slowly and failed to generate chondrocytes in vitro. The finding was in striking contrast to the mouse yolk sac showing neither adipogenic nor osteogenic potential. Together with previous knowledge, we propose that typical MSCs, like adult-type HSCs, initially develop in the AGM prior to their emergence in fetal liver and marrow. To understand the regulatory mechanisms of endothelial niche on the development of hemangioblast as well as MSC, we focused on the transforming growth factor-β (TGF-β)/Smad signaling pathway. Here, we deleted Smad4 gene, a central mediator of TGF-β signaling, specifically in endothelial cells (ECs) using the Cre–LoxP system. ECspecific Smad4 mutant mice died at embryonic day 10.5 due to cardiovascular defects including attenuated vessels sprouting and remodeling, and collapsed dorsal aorta. Noticeably, Smad4-deficient ECs demonstrated an intrinsic defect in tube formation in vitro. Furthermore, Smad4-deficient ECs dissociated away from the surrounding cells and the mutant embryos suffered from impaired development of vascular smooth muscle cells. The disturbed vascular integrity and maturation in Smad4 mutant mice was associated with altered expression of angiopoietins and downregulation of a gap junction component, connexin43. Our ongoing work is investigating whether the development of hemangioblast and MSC are defective in the mutant embryos. Cell Research (2008) 18:s54. doi: 10.1038/cr.2008.144; published online 4 August 2008