In Situ Visualization of Human MSC Engraftment in Bone Marrow: Integration into Murine Microenvironment and Interaction with Human HSC.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1283-1283
Author(s):  
Yukari Muguruma ◽  
Takashi Yahata ◽  
Hiroko Miyatake ◽  
Kiyoshi Ando ◽  
Tomomitsu Hotta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
Human Mscs ◽  
Murine Bone Marrow ◽  
Visual Evidence

Abstract Bone marrow is a complex organ system composed of two distinct lineages of cells: the hematopoietic cells and the supporting stromal cells, often referred as hematopoietic microenvironment (HME). Mesenchymal stem cells (MSCs) in bone marrow are shown to give rise to some of the components of HME, including osteoblasts, adipocytes and stromal fibroblasts in vitro, and to endothelial cells in vivo. It is a well accepted, but not definitely proven, concept that the HME provides structural niches, where dormant hematopoietic stem cells (HSCs) reside, and controls their renewal and differentiation. Although cotransplantation of human MSCs together with human HSCs resulted in increased chimerism of HSCs in animal models, existence of donor MSCs could only be detected using sensitive PCR-based analysis. Until this date, there is no physical evidence that transplanted MSCs have indeed engrafted in bone marrow and directly participated in that biological effect. In this study, we present the visual evidence for the sustained integration of human MSCs in murine bone marrow. Furthermore, we are able to delineate the physical interaction of injected human MSCs and cord blood derived CD34-positive HSCs (CBCD34). In order to assess the spatial distribution, lineage commitment and interaction of MSCs and HSCs in situ, we transplanted green fluorescent protein (GFP)-transduced MSCs and yellow fluorescent protein (YFP)-transduced CBCD34 into tibia of NOD/SCID mice. Ten weeks after intramedullary injection, longitudinal sections of mouse tibiae were made and stained with various antibodies for multicolor immunofluorescent analysis using a confocal microscope. We detected not only the existence of GFP-expressing MSCs in bone marrow, but also differentiation into several cell lineages. GFP-expressing cells exhibited phenotype and morphplogy of N-cadherin-positive bone lining osteoblasts, osteocalcin-positive osteocytes in bone, cells lining abluminal surface of vasculature, and in rare occasion, CD34 and CD31-positive endothelial cells. We then quantitatively evaluated the proportion of GFP-MSCs interacted with primitive YFP-CD34 and lineage committed YFP-CD15 and -Glycophorin-expressing cells as well as the proportion of above mentioned hematopoietic cells interacted with GFP-MSC. Approximately 50% of MSCs associated with CD34-posititive stem cells compared to only 2% and 3% of those with CD15 and Glycophorin-positive cells, respectively. It was also evident that the frequency of CD34-positive cells interacted with MSCs was significantly higher than those with CD15 and Glycophorin-positive cells. The results were consistent with a long appreciated notion that more primitive cells closely interact with hematopoietic supporting stromal cells. Furthermore, we quantitatively proved that the majority of YFP-CD34-positive HSCs were found close proximity to the bone. By transplanting GFP-MSCs together with YFP-HSCs, this study provided direct visual evidence that transplanted human MSCs engrafted in murine bone marrow and integrated into HME, which physically interacted with human HSC.

scholarly journals A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.

1991 ◽  
Vol 114 (3) ◽  
pp. 557-565 ◽  
Author(s):  
K Miyake ◽  
K Medina ◽  
K Ishihara ◽  
M Kimoto ◽  
R Auerbach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Adhesion Molecule ◽  
B Lymphocyte ◽  
Murine Bone Marrow

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.

scholarly journals Deciphering Master Gene Regulators and Associated Networks of Human Mesenchymal Stromal Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 557
Author(s):  
Elena Sánchez-Luis ◽  
Andrea Joaquín-García ◽  
Francisco J. Campos-Laborie ◽  
Fermín Sánchez-Guijo ◽  
Javier De las Rivas
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Structure ◽  
Regulatory Gene ◽  
Human Mscs ◽  
Protein Activity ◽  
Hematopoietic Stem

Mesenchymal Stromal Cells (MSC) are multipotent cells characterized by self-renewal, multilineage differentiation, and immunomodulatory properties. To obtain a gene regulatory profile of human MSCs, we generated a compendium of more than two hundred cell samples with genome-wide expression data, including a homogeneous set of 93 samples of five related primary cell types: bone marrow mesenchymal stem cells (BM-MSC), hematopoietic stem cells (HSC), lymphocytes (LYM), fibroblasts (FIB), and osteoblasts (OSTB). All these samples were integrated to generate a regulatory gene network using the algorithm ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks; based on mutual information), that finds regulons (groups of target genes regulated by transcription factors) and regulators (i.e., transcription factors, TFs). Furtherly, the algorithm VIPER (Algorithm for Virtual Inference of Protein-activity by Enriched Regulon analysis) was used to inference protein activity and to identify the most significant TF regulators, which control the expression profile of the studied cells. Applying these algorithms, a footprint of candidate master regulators of BM-MSCs was defined, including the genes EPAS1, NFE2L1, SNAI2, STAB2, TEAD1, and TULP3, that presented consistent upregulation and hypomethylation in BM-MSCs. These TFs regulate the activation of the genes in the bone marrow MSC lineage and are involved in development, morphogenesis, cell differentiation, regulation of cell adhesion, and cell structure.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

scholarly journals Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 122-131 ◽  
Author(s):  
Petter S. Woll ◽  
Julie K. Morris ◽  
Matt S. Painschab ◽  
Rebecca K. Marcus ◽  
Aimee D. Kohn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Embryonic Stem Cells ◽  
Wnt Signaling ◽  
Human Embryonic Stem Cells ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34brightCD31+Flk1+ cells and a later population of CD34dimCD45+ cells. While the CD34brightCD31+Flk1+ have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34dimCD45+ cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34brightCD31+Flk1+ cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.

scholarly journals Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 361-369 ◽  
Author(s):  
PE Funk ◽  
PW Kincade ◽  
PL Witte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Factor C

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

scholarly journals Adaptation of Marrow Osteoblast Morphology Mediated By a Hematopoietic-Derived Endosteal Cell Is Critical for Donor HSC Engraftment after BMT

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3603-3603 ◽  
Author(s):  
Kathleen Overholt ◽  
Satoru Otsuru ◽  
Victoria Best ◽  
Adam Guess ◽  
Timothy S. Olson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Diphtheria Toxin ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Hematopoietic stem cells reside in the bone marrow within specialized microenvironments designated the stem cell niche. The remarkable advances over the past decade have dramatically enhanced our perception of the niche; yet, the operative mechanisms after radioablation in preparation for bone marrow transplantation (BMT) remain poorly understood. We have previously described a profound remodeling of the bone marrow architecture after total body irradiation (TBI). This remodeling, comprised of enlarged, proliferating marrow osteoblasts and megakaryocyte migration from the central marrow space to the endosteal surface, is essential for efficient engraftment of donor cells after BMT; hence, marrow remodeling seems to represent an adaptation of the endosteal niche. To investigate whether hematopoietic cells regulate these changes, we sought to deplete all hematopoietic cells prior to TBI. We generated mice expressing the diphtheria toxin receptor (DTR) in all CD45-derived cells using the Cre/loxP model. To validate this strategy, we first crossed CD45Cre mice, where cre is expressed under the control of the endogenous promoter, with Z/RED mice which will then irreversibly express red fluorescent protein (RFP) in all cells that were derived from CD45-expressing progenitors. Surprisingly, we identified a population of RFP-expressing cells residing among osteoblasts along the endosteal and trabecular bone surfaces (designated red Bone Lining Cell, red BLC). By immunofluorescence staining, these cells lacked expression of CD45, lineage markers (Gr1, CD11b, F 4/80, CD3, B220, Ter119), and cathepsin K indicating it is not a hematopoietic cell, specifically not an osteal macrophage or osteoclast, but was unequivocally derived from CD45-expressing progenitors. We reproduced this fate map by crossing vav1Cre mice with Z/RED mice, confirming the identification and hematopoietic lineage of the red BLC. When crossed with Col2.3GFP transgenic mice, which express green fluorescent protein (GFP) in mature osteoblasts, red BLCs lacked GFP co-expression indicating it is not a generic osteoblast. Interestingly, after TBI, red BLCs markedly proliferate, but do not enlarge, in the metaphysis and epiphysis, but not in the diaphysis, coincident with the osteoblast proliferation suggesting a possible role in marrow remodeling. To pursue our original hypothesis that hematopoietic cells may regulate marrow remodeling, we treated mice expressing DTR in all CD45-derived cells and their non-expressing littermates (controls) with diphtheria toxin (DT) followed by TBI to induce marrow remodeling without the effect of CD45-derived cells. Marrow remodeling ensued; however, the characteristically enlarged endosteal osteoblasts adopted a strikingly flattened morphology (cell thickness, 8.45±0.31 vs. 3.42±0.11 μm, P<0.0001). We then used our competitive secondary transplantation assay to assess engraftment of long-term hematopoietic stem cells (HSCs) in primary recipients. Only 1 of 15 CD45-cell depleted mice engrafted HSCs compared to 10 of 15 control mice (P=0.0017) indicating a critical role of osteoblast morphology, governed by a CD45-derived cell, for donor stem cell engraftment in BMT. Megakaryocytes (Mks) and monocytes/macrophages (MMs) are the two marrow hematopoietic lineages that are recognized to survive short term after TBI and we have shown that the CD45-derived red BLC survives and proliferates after TBI. To determine if these cells regulate osteoblasts, we depleted Mks by treating Mk-specific DTR-expressing mice (generated with PF4Cre mice) with DT (>95%), and in separate cohort, MMs using clondronate (>95%). In each cohort, post-TBI marrow remodeling included the expected enlarged endosteal osteoblasts indistinguishable from controls, suggesting that neither Mks nor MMs direct the acquired osteoblast morphology. Collectively, our data indicate that enlarging of endosteal osteoblasts after marrow ablation is critical for donor cell engraftment, possibly due to altered adhesive properties for primitive hematopoietic cells. During post-TBI marrow remodeling, a CD45-derived cell that survives radioablation governs this osteoblast morphology. Our data implicate the red BLC as this key regulatory element. Understanding the red BLC will likely offer new insight into the niche and may lead to novel strategies to enhance HSC engraftment in BMT. Disclosures No relevant conflicts of interest to declare.

New Evidence That the Bioactive Lipid Ceramide-1-Phosphate (C1P) Is a Potent Chemoattractant for Mesenchymal Stromal Cells (MSC), Endothelial Progenitor Cells (EPCs) and Very Small Embryonic-Like Stem Cells (VSELs), Demonstrating Its Potential Involvement in Tissue/Organ Repair and Angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2387-2387 ◽  
Author(s):  
ChiHwa Kim ◽  
Rui Liu ◽  
Magda Kucia ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Tube Formation ◽  
Bioactive Lipids ◽  
Cytokines And Chemokines ◽  
Organ Regeneration ◽  
Organ Repair ◽  
Ceramide 1

Abstract Abstract 2387 Background: Ceramide-1-phosphate (C1P), like its related derivative sphingosine-1-phosphate (S1P), is derived from sphingolipids, which are important components of cell membranes. In contrast to S1P, which is secreted as a signaling molecule from intact cells, C1P is released from cells that are damaged and “leaky”. Unlike ceramide (which is often pro-apoptotic), C1P is anti-apoptotic and, as we have demonstrated, does not impair the clonogenicity of hematopoietic progenitors (Leukemia 2011, doi: 10.1038/leu.2011.185) and promotes their migration through an unknown receptor-initiated signaling pathway that is pertussis toxin-sensitive and therefore likely to involve a Gai protein-coupled seven-transmembrane-spanning receptor. Hypothesis: Taking into consideration the anti-apoptotic effects of C1P and the fact that it is released in damaged tissues, we have hypothesized that C1P plays an important and under-appreciated role in tissue/organ regeneration. Materials and Methods: To better address the potential role of C1P in this process we i) employed the Transwell system and FACS analysis to evaluate chemotactic responsiveness of stem cells involved in tissue and organ regeneration, including CD31+CD45−Lin− mesenchymal stroma cells (MSCs), Sca-1+CD51+CD45-Lin−CD31− endothelial progenitor cells (EPCs) and Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs), ii) measured the C1P level in damaged tissues (BM conditioned for transplantation, myocardium after heart infarct, and chemically damaged liver), iii) studied the activation of intracellular signaling pathways related to cell migration/adhesion (MAPKp42/44, AKT, and p38) in bone marrow stromal cells and umbilical cord blood-derived endothelial cells (HUVECs) in response to C1P stimulation, iv) evaluated the effect of C1P on adhesion and migration of stromal fibroblasts and endothelial cells, v) evaluated the influence of C1P on tube formation by HUVECs, and v) evaluated the effect of C1P stimulation on secretion of cytokines and chemokines by bone marrow stromal cells. All these effects were studied in parallel in comparison to other bioactive lipids (S1P, lysophosphatidic acid [LPA], and lysophosphatidylocholine [LPC]), as well as directly compared to the effects of known chemottractants of stem cells, bone marrow (BM)-derived fibroblasts, and endothelial cells (SDF-1, HGF, VEGF, and FGF-2). Results: We observed that C1P is upregulated in damaged tissues and strongly chemoattracts stem cells from BM that are potentially involved in tissue/organ regeneration (VSELs, MSCs, and EPCs). In chemotactic assays performed on expanded BM-derived fibroblasts and HUVECs, C1P had a similar strong chemotactic effect as S1P, but stronger than other bioactive lipids (LPA and LPC). C1P strongly stimulated phosphorylation of MAPKp42/44 and AKT in HUVECs and MAPKp42/44 in BM-derived fibroblasts. Interestingly, compared to all bioactive lipids tested in this study, C1P most strongly stimulated tube formation by HUVECs and stimulated secretion from BM-derived fibroblasts of several cytokines and chemokines, including stromal derived factor-1 (SDF-1). Conclusions: Our data demonstrate, for the first time, that C1P is a, potent bioactive lipid released from damaged cells that plays an important novel role not only in homing of HSPCs to BM, as we demonstrated recently, (Leukemia 2011, doi: 10.1038/leu.2011.185) but also directs migration and activates stem cells that are involved in repair of other damaged organs and tissues. We also envision that modulation of C1P signaling may turn out to be a viable strategy for tissue/organ repair and this is currently tested in our laboratory. Disclosures: No relevant conflicts of interest to declare.

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells from Induced Pluripotent Stem Cells through Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3866-3866
Author(s):  
Masao Tsukada ◽  
Satoshi Yamazaki ◽  
Yasunori Ota ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Teratoma Formation

Abstract Introduction Generation of engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has long been thought an ultimate goal in the field of hematology. Numerous in vitro differentiation protocols, including trans-differentiation and forward programming approaches, have been reported but have so far failed to generate fully functional HSCs. We have previously demonstrated proof-of-concept for the in vivo generation of fully functional HSCs from induced PSCs (iPSCs) through teratoma formation (Suzuki et al., 2013). However, this method is time-consuming (taking over two months), HSCs are generated at low frequencies, and additionally require co-injection on OP9 stromal cells and SCF/TPO cytokines. Here, we present optimization of in vivo HSC generation via teratoma formation for faster, higher-efficiency HSC generation and without co-injection of stromal cells or cytokines. Results First, we screened reported in vitro trans-differentiation and forward programming strategies for their ability to generate HSCs in vivo within the teratoma assay. We tested iPSCs transduced with the following dox-inducible TF overexpression vectors: (1) Gfi1b, cFOS and Gata2 (GFG), which induce hemogenic endothelial-like cells from fibroblast (Pereira et al.,2013); (2) Erg, HoxA9 and Rora (EAR), which induce short-term hematopoietic stem/progenitor cell (HSPC) formation during embryoid body differentiation (Doulatov et,al., 2013); and (3) Foxc1, which is highly expressed the CAR cells, a critical cell type for HSC maintenance (Oomatsu et al.,2014). We injected iPSCs into recipient mice, without co-injection of stromal cells or cytokines, and induced TF expression after teratoma formation by dox administration. After four weeks, GFG-derived teratomas contained large numbers of endothelial-like and epithelial-like cells, and importantly GFG-derived hematopoietic cells could also be detected. EAR-teratomas also generated hematopoietic cells, although at lower frequencies. By contrast, hematopoietic cells were not detected in control teratomas or Foxc1-teratomas. Through use of iPSCs generated from Runx1-EGFP mice (Ng et al. 2010), and CUBIC 3D imaging technology (Susaki et al. 2014), we were further able to demonstrate that GFG-derived hematopoietic cells were generated through a haemogenic endothelium precursor. Next, we assessed whether HSPC-deficient recipient mice would allow greater expansion of teratoma-derived HSCs. This was achieved by inducing c-kit deletion within the hematopoietic compartment of recipient mice (Kimura et al., 2011) and resulted in a ten-fold increase in the peripheral blood frequency of iPSC-derived hematopoietic cells. We further confirmed similar increases in iPSC-derived bone marrow cells, and in vivo HSC expansion, through bone marrow transplantation assays. Finally, we have been able to shorten the HSC generation time in this assay by five weeks through use of transplantable teratomas, rather than iPSCs. Conclusions We have demonstrated that GFG-iPSCs induce HSC generation within teratomas, via a hemogenic endothelium precursor, and that use of HSPC-deficient recipient mice further promotes expansion of teratoma-derived HSCs. These modifications now allow us to generate engraftable HSCs without co-injection of stromal cells or cytokines. Additionally, use of transplantable teratomas reduced HSC generation times as compared with the conventional assay. These findings suggest that our in vivo system provides a promising strategy to generate engraftable HSCs from iPSCs. Disclosures No relevant conflicts of interest to declare.

Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic Stem Cells and the Granulopoietic Differentiation of More Mature Progenitors

Stem Cells ◽  
2005 ◽  
Vol 23 (10) ◽  
pp. 1626-1633 ◽  
Author(s):  
Frédérique Hubin ◽  
Chantal Humblet ◽  
Zakia Belaid ◽  
Charles Lambert ◽  
Jacques Boniver ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

scholarly journals Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 361-369 ◽  
Author(s):  
PE Funk ◽  
PW Kincade ◽  
PL Witte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Factor C

Abstract In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

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