Bonr Marrow Reconstitution from Mouse Embryonic Stem Cell-Derived Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4145-4145
Author(s):  
Momoko Yoshimoto ◽  
Chang Hsi ◽  
Katsutsugu Umeda ◽  
Midori Iida ◽  
Toshio Heike ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Stromal Cell ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Scid Mice ◽  
Pcr Analysis ◽  
Facs Analysis ◽  
Cd34 Cells

Abstract Differentiation of embryonic stem (ES) cells in vitro yield different kinds of hematopoietic progenitors including primitive and definitive hematopoietic cells. It has been reported that HOXB4 induction enable york sac (YS) cells and embryoid body-derived cells to engraft in the irradiated adult mice, however, since the characteristic of ES-derived transplantable cells is not clear, generating hematopoietic stem cells (HSCs) in vitro still remains to be resolved. We previously reported the generation of definitive HSCs from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (Matsuoka, et al; Blood 2001) Co-cultureing on AGM-S3, these YS cells and PS-Sp cells acquired the reconstituting potential of adult irradiated mice. Here we intended to make HSCs using this stromal cells. We differentiated ES cells labeled with GFP on OP9 stromal cell, which is supportive for Hematopoietic differentiation. After 4 days, we sorted Flk1+ cells, which is considered as a marker of hemangilblasts, and transferred them onto A-9, subline of AGM-S3, or OP9 stromal cell layer with cytokines. After several days incubation, we examined the emergence of CD34+ c-kit+ cells and colony forming ability of CD34+ or CD34− cells. CD34+ cells contained more CFU-Mix than CD34− cells. When compared on A-9 or OP9, cultured cells on OP9 contained more CFU activity than on A-9. We sorted and cultured CD34+ c-kit+ cells on OP9 for 7–10 days, and confirmed Ter119+, Gr-1+, or Mac-1+ cells differentiated from CD34+ c-kit+ cells by FACS analysis. Next, we cultured Flk1+ cells on A-9 or OP9 for 7–15 days and transplanted all the collected cells into 2.4Gy irradiated NOD-SCID mice. After 3 months after transplantation, FACS analysis showed no GFP+ cells in the recipient BM. However, PCR analysis detected donor derived DNAs in BM when Flk1+ cells were cocultured on A-9. We next transplanted 1×104 of CD34+ CD45+ or CD34+ CD45− cells from Flk1+ cells cocultured on A-9 or OP9 into 2.4 Gy irradiated NOD-SCID mice. PCR analysis revealed donor derived DNAs in mice transplanted with CD34+ CD45+ cells on A-9 and with CD34+ CD45− cell on OP9. These data suggested that CD34+ cells differentiated from Flk1+ cells have powerful hematipoietic activity and showed different potential cultured between on A-9 and on OP9.

Human embryonic stem cell–derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 617-626 ◽  
Author(s):  
Maxim A. Vodyanik ◽  
Jack A. Bork ◽  
James A. Thomson ◽  
Igor I. Slukvin
Keyword(s):  
Stem Cell ◽  
Stromal Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hes Cells

AbstractEmbryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell–derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase–positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

scholarly journals Excessive Reactive Iron Impairs Hematopoiesis by Affecting Both Immature Hematopoietic Cells and Stromal Cells

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 226 ◽  
Author(s):  
Hirokazu Tanaka ◽  
J. Espinoza ◽  
Ryosuke Fujiwara ◽  
Shinya Rai ◽  
Yasuyoshi Morita ◽  
...  
Keyword(s):  
Iron Overload ◽  
Stromal Cells ◽  
Es Cells ◽  
Genetic Disorders ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
The Body ◽  
Ferrous Ammonium Sulfate ◽  
Excess Iron

Iron overload is the accumulation of excess iron in the body that may occur as a result of various genetic disorders or as a consequence of repeated blood transfusions. The surplus iron is then stored in the liver, pancreas, heart and other organs, which may lead to chronic liver disease or cirrhosis, diabetes and heart disease, respectively. In addition, excessive iron may impair hematopoiesis, although the mechanisms of this deleterious effect is not entirely known. In this study, we found that ferrous ammonium sulfate (FeAS), induced growth arrest and apoptosis in immature hematopoietic cells, which was mediated via reactive oxygen species (ROS) activation of p38MAPK and JNK pathways. In in vitro hematopoiesis derived from embryonic stem cells (ES cells), FeAS enhanced the development of dysplastic erythroblasts but inhibited their terminal differentiation; in contrast, it had little effect on the development of granulocytes, megakaryocytes, and B lymphocytes. In addition to its directs effects on hematopoietic cells, iron overload altered the expression of several adhesion molecules on stromal cells and impaired the cytokine production profile of these cells. Therefore, excessive iron would affect whole hematopoiesis by inflicting vicious effects on both immature hematopoietic cells and stromal cells.

scholarly journals Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1297-1305 ◽  
Author(s):  
N Uchida ◽  
J Combs ◽  
S Chen ◽  
E Zanjani ◽  
R Hoffman ◽  
...  
Keyword(s):  
Stromal Cell ◽  
Biological Activities ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
In Utero ◽  
Rhodamine 123 ◽  
Differentiation Potential ◽  
Cd34 Cells

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Natural Killer and B-Lymphoid Potential in CD34+ Cells Derived From Embryonic Stem Cells Differentiated in the Presence of Vascular Endothelial Growth Factor

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2283-2295 ◽  
Author(s):  
Naoki Nakayama ◽  
Inghwa Fang ◽  
Gary Elliott
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Natural Killer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cytotoxic Lymphocytes ◽  
Vascular Endothelial ◽  
Cd34 Cells ◽  
Endothelial Growth Factor

Abstract Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34−lymphocytes: CD3− cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+intermediate, on which VEGF may play an important role.

Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 495-495
Author(s):  
Ryo Kurita ◽  
Erika Sasaki ◽  
Takashi Hiroyama ◽  
Tomoko Yokoo ◽  
Yukoh Nakazaki ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Preclinical Studies ◽  
Es Cell ◽  
Hematopoietic Stem

Abstract Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.

FLT3 Immunotherapy Can Eliminate Engraftment of ALL Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 869-869
Author(s):  
Obdulio Piloto ◽  
Bao Nguyen ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
David Huso ◽  
...  
Keyword(s):  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Facs Analysis ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Monocyte and Fibroblast Directed Generation of a Marrow Vascular Niche

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3602-3602
Author(s):  
Surya S Kotha ◽  
Kiet T Phong ◽  
Amie Adams ◽  
Brian Hayes ◽  
Meredith A Roberts ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Cell Trafficking ◽  
Stromal Microenvironment ◽  
Cd34 Cells ◽  
The Matrix ◽  
Stem Cell Trafficking

Abstract Hematopoietic cells dynamically interact with their surrounding microenvironment during their residence, maturation and differentiation. Individual marrow components have been isolated and studied in 2D in vitro cultures, yet their functional contributions to a complete niche are not fully understood. In vivo studies in mouse models are complex, and the inaccessibility of marrow architecture has precluded systematic analysis of each component. Here, we employ an in vitro 3D microfluidic vascular system to study the effect of microenvironmental cues on cell trafficking in an engineered hematopoietic niche. Our system allows for control of 3D geometric cues, hydrodynamic flow, multi-cellular compositions, and cellular matrix remodeling by combining soft lithography and injection modeling in type I collagen gel (Zheng et al. PNAS 2012). Endothelial cells perfused through the embedded microfluidic network form a confluent, patent endothelium within the collagen. Incorporating hematopoietic cells and stromal cells into the extravascular collagen space allows us to visualize how cells interact with vasculature during culture. First, we developed a marrow stromal microenvironment to understand how stromal cells modify the marrow microvascular environment, by incorporating two different human marrow-derived stromal fibroblast cell lines (HS27a and HS5) in the extravascular space surrounding the vessels (Fig 1A). HS27a and HS5 cells created distinct vascular microenvironments by secreting divergent cytokine profiles: stem cell niche-associated and inflammatory cytokines, respectively. Both stromal lines modified the vascular phenotype by reducing endothelial expression of vWF and junctional proteins. In particular, co-culture with HS5 increased expression of inflammatory markers on the endothelium. Next, we evaluated the function of this modified vasculature on hematopoietic stem cell trafficking through the vessels. When CD34+ cells alone were perfused through the microvessels, they adhered on the vessel wall and migrated into the matrix. The HS27a and HS5-induced microenvironment did not significantly change trafficking behavior (Fig 1B, C). When monocytes were perfused through the microvessels, they also adhered on the vessel wall and migrated into the matrix. Monocytes are a critical part of the marrow space, and are known to modify the endothelium and stromal microenvironment. When CD34+ cells were perfused through the vessels 24 hours after monocytes, crosstalk shifted trafficking patterns of both cell types and led to increased adhesion and migration within the HS5- and HS27a-modified vessels (Fig 1D). To explore the competent marrow niche in vitro, we further examined hematopoietic cell trafficking from the extravascular space into the circulation by embedding cells from fresh human bone marrow screens within the collagen matrix. We found this cell fraction contained a mixed population of hematopoietic and stromal cells, and could be cultured in the microvascular niche for at least two weeks. Scanning electron microscopy showed various types of marrow cells residing in both the abluminal and luminal side of the endothelium (Fig 1E-G). Throughout culture with continuous vascular perfusion, we collected the media flow-through and identified different hematopoietic cell populations released from the matrix into the circulation over the course of two weeks. Specifically, we identified CD34+ hematopoietic progenitor cells along with megakaryocyte, erythroid, lymphoid, and myeloid lineage cells by flow cytometry analysis. In summary, we developed an in vitro 3D microvascular marrow niche and gained insight into hematopoietic cell trafficking between the stroma and the circulation. By guiding the interplay of heterogeneous cell populations, we have demonstrated the capacity to define distinct microenvironment spaces. This platform shows promise for long term culture of a whole marrow population and for the ex vivo generation of hematopoietic cells. Further development of this 3D marrow niche will allow us to better understand the complexities mediating stem cell trafficking, residence, proliferation, mobilization, and differentiation in both health and disease. Disclosures No relevant conflicts of inteqerest to declare.

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