Human CD8+/TCR−/CD56dim/- Facilitating Cells Facilitate Homing and Engraftment of Human HSC in Vivo

Abstract Abstract 4093 Hematopoietic stem cell (HSC) transplantation has become a common procedure for treatment of hematopoietic malignancies and autoimmune disease. Despite significant advances in HSC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain limitations to application in the clinic. However, these risks can be overcome through less toxic nonmyeloablative conditioning and cell depletion strategies to remove GVHD causing-cells while retaining engraftment enhancing-tolerogeneic cells. We were the first to discover CD8+/TCR− graft facilitating cells (FC) in mouse bone marrow. The addition of as few as 30,000 FC to 10,000 HSC significantly enhances engraftment of HSC in allogeneic recipients without causing GVHD. FC also potently enhance engraftment of limiting numbers of syngeneic HSC. Human CD8+/TCR- FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (mPBMC). In the CD8+/TCR- FC, 48% of cells expressed CD3ε+, 43% were FoxP3+, 43% were CD11c+, 19% were CD19+, and 30% were HLA-DR+. Approximately 55% of FC are also CD56dim/-, and the remaining population is CD56bright. The morphology of human CD8+/TCR− FC with Wright-Giemsa staining under light microscopy suggested that the human FC population is heterogeneous. Here we evaluated if human FC enhance human HSC or progenitor homing to bone marrow of NOD/SCID/IL-2rγnull (NSG) mouse recipients. CD45+CD34+ HSC and CD8+/TCR−/CD56dim/-FC were sorted from mPBMC. NSG recipients were conditioned with 1100 cGy of total body irradiation (TBI). 24 hours after TBI, 100,000 HSC with or without 300,000 CD8+/TCR−/CD56dim/- FC were transplanted into conditioned NSG recipients. Recipients were euthanized 16 hours after transplantation. Bone marrow was harvested from femurs and tibias of recipients and plated in Colony Forming Culture (CFC) Assays. Recipients of HSC plus FC generated significantly more colony formation (colonies = 110) compared with HSC alone (colonies = 65) (P = 0.011), suggesting that CD8+/TCR−/CD56dim/- FC enhanced homing of HSC or progenitors to bone marrow. To test if human CD8+/TCR−/CD56dim/- FC facilitate engraftment of human HSC in NSG mice, 300,000 CD8+/TCR−/CD56dim/- FC were mixed with 100,000 HSC and transplanted into NSG recipient mice conditioned with 325 cGy TBI. Mice that received HSC alone served as controls. At 30 days after transplantation, PBL typing showed that 34% (10 of 29) recipients of HSC alone engrafted. In contrast, 78% of recipients (n = 23) of HSC plus CD8+/TCR−/CD56dim/- FC engrafted, and donor chimerism in PB was 1.1% ± 0.8% and 4.1% ± 1.3% (P <0.05), respectively. At 6 months after transplantation, NSG recipients of HSC + CD8+/TCR−CD56dim/- FC exhibited persistent donor chimerism in PB (9.1% ± 6% vs. 3.8% ± 3.5%) (P <0.05) and significantly higher levels of donor chimerism in spleen (26.3% ± 11.8% vs. 12.3% ± 9.8%) (P <0.05) and BM (11.6% ± 4.8% vs. 2.9% ± 1.3%) (P <0.05) compared to recipients of HSC alone. Our data indicate that CD8+/TCR−/CD56dim/- FC facilitate homing of human HSC or progenitors and enhance engraftment of human HSC in NSG recipient mice. Disclosures: Bozulic: Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

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Human CD8+/TCR−/CD56dim/− facilitating Cells Enhance Engraftment of HSC In NOD/SCID/IL2rγnull Recipients.

Blood ◽

10.1182/blood.v116.21.3709.3709 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3709-3709

Author(s):

Yiming Huang ◽

Mary J Elliott ◽

Deborah M Ramsey ◽

Thomas Miller ◽

Larry D Bozulic ◽

...

Keyword(s):

Cell Population ◽

Mononuclear Cells ◽

No Donor ◽

Peripheral Blood Mononuclear ◽

Hematopoietic Stem ◽

Donor Chimerism ◽

Equity Ownership ◽

Total Body

Abstract Abstract 3709 CD8+/TCR− graft facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC). They are distinct from HSC since they do not generate multilineage engraftment when infused alone, nor do they form colonies in vitro. The function of human FC remains to be defined. Here, we phenotypically characterized the human CD8+/TCR− FC subpopulations and evaluated their function in vivo and in vitro. Human CD8+/TCR− FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (PBMC). Approximately 55% of FC express CD56dim/−, and the remaining population is CD56bright. In the CD8+/TCR−/CD56bright cell population, approximately 65% of cells express CD11c+ and 67% of cells express CD11b+. In CD8+/TCR−/CD56dim/− cell population, the majority of cells express CD3ε+ (80%), 17% were CD11c+, 16% were CD19+, 14% were CD11b+, 11% were CD123+, and 30% were HLA-DR+. To evaluate whether human CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo, we transplanted 100,000 HSC alone or plus 300,000 CD8+/TCR−/CD56dim/− FC into NOD/SCID/IL2rγnull (NSG) recipient mice conditioned with 325 cGy of total body irradiation. At 30 days after transplantation, 8 of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of recipients (n = 16) receiving HSC plus CD8+/TCR−/CD56dim/− FC engrafted, and donor lymphocyte and monocyte chimerism in PB was 0.53% ± 0.16% and 3.93% ± 1.28%, respectively. At 6 months after transplantation, NSG recipients of HSC alone lost donor chimerism in PB and no donor cells were detected in spleen and BM. In contrast, NSG recipients of HSC + CD8+/TCR−CD56dim/− FC exhibited durable donor chimerism in PB and showed significantly higher levels of donor chimerism in spleen (13%) and BM (9.43%) compared to recipients of HSC alone. To evaluate the function of CD8+/TCR−/CD56dim/− FC in vitro, HSC were incubated with CD8+/TCR−CD56dim/− FC for 18 hrs and then cultured in methylcellulose for 14 days in colony-forming cell assay. HSC plus CD8+/TCR−CD56dim/− FC generated significantly more colonies compared with HSC alone (P = 0.0038), suggesting that human CD8+/TCR−/CD56dim/− FC have a direct effect on the clonogenicity of HSC (Figure). In summary, our data indicate that (1) human CD8+/TCR− FC are a heterogeneous cell population; (2) CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo and promote HSC clonogenicity in vitro; and (3) recipients of CD8+/TCR−/CD56dim/− FC maintain a durable donor chimerism in BM and spleen. Disclosures: Bozulic: Regenerex, LLC: Employment. King:Regenerex, LLC: Employment, Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.

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Effective Erythropoiesis from Human iPSC-Derived RBC in Immunodeficient Mice

Blood ◽

10.1182/blood-2020-138606 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-42

Author(s):

Jiusheng Deng ◽

Moira M. Lancelot ◽

Ryan Jajosky ◽

Kristin Deeb ◽

Natia Saakadze ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Publicly Traded ◽

Peripheral Blood Mononuclear ◽

Immunodeficient Mice ◽

Nsg Mice ◽

Human Ipsc

Transfusion of red blood cells (RBCs) was the earliest developed form of cell therapy and is still a highly effective life-saving treatment for many patients. Induced pluripotent stem cells (iPSC) can differentiate into RBCs (iPSC-RBCs) and may provide a novel source for blood transfusion and a cellular model for erythroid differentiation. Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. Human iPSC were generated from peripheral blood mononuclear cells of healthy donors by transfection of plasmids containing OCT4, SOX2, MYC, KLF4 and BCL-XL genes. iPSC lines expressed TRA-1-60, SSEA4 and Nanog markers, and showed a normal karyotype. iPSCs were induced to differentiate along the erythroid lineage using a 3-stage culture system requiring 33 days. At the end of the culture period, iPSC-RBCs were CD34-CD235a+CD41+CD43+CD71low; about 10% of cells were enucleated (CD235a+DRAQ5-). iPSC-RBCs were harvested and transfused into immunodeficient NSG mice which had been pretreated with clodronate liposomes and cobra venom factor (CL/CVF). CL/CVF treatment of NSG mice markedly promoted the survival of transfused human iPSC-RBC in vivo, which could be detected with anti-human CD235a antibodies for at least 7 days, although the numbers progressively decreased with time. Interestingly, a large number of transfused iPSC-derived cells homed to bone marrow of NSG mice. In NSG mice that were repetitively treated with CL/CVF every 3 days, nucleated iPSC-derived cells were still detectable in the bone marrow 4 weeks after transfusion. Furthermore, at 3 weeks after transfusion, human iPSC-RBCs reappeared in the peripheral circulation. These circulating iPSC-RBCs were > 90% enucleated and were present at levels more than 4-fold higher than at 1 hour after transfusion. These results suggest that iPSC-RBCs which homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into the peripheral blood. The results offer a new model using human peripheral blood iPSC and CL/CVF-treated NSG mice to investigate the development of human erythroid cells in vivo. Disclosures Jajosky: Biconcavity Inc.: Other: CEO and partial owner; BioMarin Pharmaceuticals: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company.

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Recombinant TAT-HOXB4 Protein Promotes Ex Vivo Expansion of Primitive Human Hematopoietic Cells.

Blood ◽

10.1182/blood.v104.11.2855.2855 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2855-2855

Author(s):

Gorazd Krosl ◽

Marie-Pier Giard ◽

Jana Krosl ◽

R. Keith Humphries ◽

Guy Sauvageau ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Cell Populations ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.

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Prospective Isolation of Human Eosinophil-Committed Progenitors.

Blood ◽

10.1182/blood.v108.11.1285.1285 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1285-1285

Author(s):

Yasuo Mori ◽

Hiromi Iwasaki ◽

Goichi Yoshimoto ◽

Aki Okeda ◽

Toshihiro Miyamoto ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Inflammatory Diseases ◽

Hypereosinophilic Syndrome ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Human Eosinophil ◽

Murine Bone Marrow

Abstract Eosinophils play an important role in the pathogenesis of allergic reactions or chronic inflammatory diseases by releasing various types of cytokines and chemical mediators. Recently, we have identified murine eosinophil-committed progenitors (mEoPs) in mouse bone marrow. The expression of receptor for IL-5, a critical cytokine for proliferation and differentiation of eosinophils, was a key marker to isolate mEoPs: mEoP was IL-5Ra+Lineage(lin)-CD34+c-Kitlow population in murine bone marrow (J Exp Med.201, 1891ndash;7, 2005). Here we report that EoPs are prospectively isolatable also in human bone marrow. We analyzed the expression of human IL-5Ra in human stem and progenitor populations, and found that a fraction of common myeloid progenitor (CMP; lin-CD34+CD38+CD45RA-IL-3Ra+) population expressed hIL-5Ra on their surface by using anti-human IL-5Ra monoclonal antibodies. IL-5Ra protein and mRNA were undetectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), megakaryocyte/erythrocyte progenitors (MEPs; lin-CD34+CD38+CD45RA-IL-3Ra-), or granulocyte/monocyte progenitors(GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS and RT-PCR, respectively. The IL-5Ra+ cells within the CMP fraction constituted only ~0.04% of steady-state bone marrow mononuclear cells, and gave rise only to pure eosinophil colonies. Thus we termed this population as human EoP (hEoP). Both HSCs and the IL-5Ra- fraction of CMPs gave rise to IL-5Ra+ hEoPs in vitro in the presence of IL-3 and GM-CSF, while MEPs or GMPs never generated hEoPs, indicating that human eosinophil pathway diverges at the CMP stage, and that the eosinophil potential was lost at the GMP or MEP stage. Accordingly, the human eosinophil pathway is different from that in murine hematopoiesis where mEoPs develop from the GMP stage. Strikingly, the number of hEoPs in the bone marrow of patients with hypereosinophilic syndrome was significantly (~4-fold) increased as compared to that in normal bone marrow, suggesting that hEoP represents a critical stage for eosinophilia in vivo. Thus, the hEoP is an attractive candidate for therapeutic target in eosinophil-related allergic and inflammatory disorders. This population might also be very useful to study the molecular mechanism of human eosinophil development.

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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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Interleukin-2 production and response to interleukin-2 by peripheral blood mononuclear cells from patients after bone marrow transplantation: II. Patients receiving soybean lectin-separated and T cell-depleted bone marrow

Blood ◽

10.1182/blood.v70.5.1595.1595 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1595-1603 ◽

Cited By ~ 23

Author(s):

K Welte ◽

CA Keever ◽

J Levick ◽

MA Bonilla ◽

VJ Merluzzi ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

Abstract The ability of peripheral blood mononuclear cells (PBMC) to produce and respond to interleukin-2 (IL-2) was evaluated in 50 recipients of HLA- identical bone marrow (BM) depleted of mature T cells by soybean agglutination and E rosetting (SBA-E-BM). In contrast to our previous findings in recipients of unfractionated marrow, during weeks 3 to 7 post-SBA-E-BM transplantation (BMT), PBMC from the majority of patients spontaneously released IL-2 into the culture medium. This IL-2 was not produced by Leu-11+ natural killer cells, which were found to be predominant in the circulation at this time, but by T11+, T3+, Ia antigen-bearing T cells. The IL-2 production could be enhanced by coculture with host PBMC frozen before transplant but not by stimulation with mitogenic amounts of OKT3 antibody, thus suggesting an in vivo activation of donor T cells or their precursors by host tissue. Spontaneous IL-2 production was inversely proportional to the number of circulating peripheral blood lymphocytes and ceased after 7 to 8 weeks post-SBA-E-BMT in most of the patients. In patients whose cells had ceased to produce IL-2 spontaneously or never produced this cytokine, neither coculture with host cells nor stimulation with OKT3 antibody thereafter induced IL-2 release through the first year posttransplant. Proliferative responses to exogenous IL-2 after stimulation with OKT3 antibody remained abnormal for up to 6 months post-SBA-E-BMT, unlike the responses of PBMC from recipients of conventional BM, which responded normally by 1 month post-BMT. However, the upregulation of IL- 2 receptor expression by exogenous IL-2 was found to be comparable to normal controls when tested as early as 3 weeks post-SBA-E-BMT. Therefore, the immunologic recovery of proliferative responses to IL-2 and the appearance of cells regulating in vivo activation of T cells appear to be more delayed in patients receiving T cell-depleted BMT. Similar to patients receiving conventional BMT, however, the ability to produce IL-2 after mitogenic stimulation remains depressed for up to 1 year after transplantation.

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Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽

10.1182/blood-2002-12-3893 ◽

2003 ◽

Vol 101 (12) ◽

pp. 4680-4686 ◽

Cited By ~ 144

Author(s):

Kent W. Christopherson ◽

Scott Cooper ◽

Hal E. Broxmeyer

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mouse Bone Marrow ◽

Peptidase Activity ◽

Hematopoietic Stem ◽

Migratory Response ◽

Stromal Cell Derived Factor ◽

Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

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Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Blood ◽

10.1182/blood.v108.11.1653.1653 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1653-1653

Author(s):

Nobuyuki Onai ◽

Aya Onai ◽

Markus G. Manz

Keyword(s):

Bone Marrow ◽

Steady State ◽

Type I Interferon ◽

Receptor Expression ◽

Mouse Bone Marrow ◽

Type I ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Gm Csf

Abstract Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions. To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity. In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

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Rapid and Irreversible Alteration of the Ability of Hematopoietic Stem Cells To Execute Both Symmetric and Asymmetric Self-Renewal Divisions by Exposure to Reduced Steel Factor Concentrations with No Effect on Their Survival or Mitogenesis.

Blood ◽

10.1182/blood.v108.11.684.684 ◽

2006 ◽

Vol 108 (11) ◽

pp. 684-684

Author(s):

David G. Kent ◽

Brad Dykstra ◽

Connie J. Eaves

Keyword(s):

Bone Marrow ◽

Adult Mouse ◽

Single Cells ◽

Mouse Bone Marrow ◽

Self Renewal ◽

Hematopoietic Stem ◽

Steel Factor ◽

High Concentrations

Abstract Hematopoietic stem cells (HSCs) are present in the marrow of adult mice at a frequency of 1/104, as measured by limiting dilution transplantation assays for individual cells that produce lymphoid (B and T) as well as myeloid (GM) cells for at least 4 months in irradiated recipients. HSCs thus defined can be reproducibly isolated in the CD45midlin−Rho−SP fraction of adult mouse bone marrow at a purity of >30%. In mice, mutations in c-kit, the receptor for Steel factor (SF) lead to substantial reductions in the adult HSC population. In vitro, SF has been identified as a potent regulator of HSC self-renewal divisions. High concentrations of SF in combination with IL-11 allow adult HSCs to divide with a net 2–4 fold expansion in HSC numbers after 10 days and low concentrations of SF result in loss of HSC activity. To investigate the cellular mechanisms underlying these different outcomes, we cultured 114 CD45midlin−Rho−SP adult mouse bone marrow cells in single cell cultures containing serum-free medium + 20 ng/ml IL-11 and either 300 or 10 ng/ml of SF. Each culture was then examined every 4–6 hr. The kinetics of division of these cells under both conditions was identical with completion of the 1st division occurring between 22–68 hr. During that time none of the input cells died (<1%). After 10 days of culture, during which time all input cells divided at least 5 times (>50 cells), the HSC content of pooled clones (as measured by in vivo transplantation assays) was found to be >10-fold higher in the clones generated under high vs. low SF conditions (p<0.05). To characterize the types of self-renewal divisions undertaken, 9 doublets generated under the high SF condition were harvested between 4 and 8 hr after they underwent their 1st division and then each of the daughters was injected into a separate irradiated mouse. Analysis of the 18 mice showed that for one of the input cells both daughters were HSCs (evidence of a symmetric self-renewal division) and for 3 more, only one of the 2 daughters was an HSC (evidence of an asymmetric self-renewal division). In contrast no daughter HSCs were identified when 6 doublets produced under the low SF condition were assayed. To determine whether the loss of HSC activity under low SF conditions was a pre- or post-mitotic event, additional in vivo HSC assays were performed on cells harvested from individual wells after 8, 16 and 96 hours of incubation. The results revealed no change in the proportion of wells with either low or high concentrations of SF that contained HSCs after 8 hr of incubation (10/36 positive mice injected with starting single cells and 5/17 (low SF) vs. 6/17 (high SF) positive mice injected with 8-hr single cells, respectively). However, a significant difference (p<0.01) was seen after 96 hr (5/35 vs. 2/43 positive mice, respectively) and, after only 16 hr, before a first mitosis was seen under either condition, a decline in HSCs was apparent under the low SF condition (4/15 vs. 1/15 positive mice injected with cells from the high vs. low SF condition). Together, these studies indicate that HSC exposure to different SF concentrations can rapidly and irreversibly alter the ability of HSCs to execute symmetric as well asymmetric self-renewal divisions in vitro.

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Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Blood ◽

10.1182/blood.v118.21.4168.4168 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4168-4168

Author(s):

Satiro N. De Oliveira ◽

Francesca Giannoni ◽

Cinnamon Hardee ◽

Arineh Sahaghian ◽

Laurence J N Cooper ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Progenitor Cells ◽

Malignant Cells ◽

Hematopoietic Stem ◽

Effector Cells ◽

Nsg Mice ◽

Novel Approach ◽

B Lineage

Abstract Abstract 4168 Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures: No relevant conflicts of interest to declare.

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