Timing of Mesenchymal Stem Cell Co-Transplantation and Site of Bone Engraftment Determine Levels of Hematopoietic Engraftment and Contribution of HSC to the Bone Niche

Abstract Abstract 940 We and others have previously demonstrated that the co-transplantation of marrow-derived stromal cells (MSC) with hematopoietic stem cells (HSC) results in higher levels of engraftment and acceleration of the rate of appearance of donor derived hematopoietic cells in the peripheral blood (PB) following transplantation. Possible explanations for this effect are MSC immunomodulatory properties and/or the ability of MSC to produce factors which support the transplanted HSC or prevent them from undergoing apoptosis. Here, we hypothesized that transplanted MSC are able to engraft within the recipient's bone marrow and integrate into vascular and/or osteoblastic niches to selectively create HSC donor optimized sites, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered intra-peritoneally 1.4×10^5 CD34+ cells transduced with a lentiviral vector encoding eGFP, (eGFP-CD34+) in combination with 2.5×10^5 same donor MSC transduced with a lentiviral vector encoding mKate (mKateMSC) (n=4). Another group of animals (n=4) received 2.5×10^5 mKateMSC, 3 days prior to transplantation of same donor 1.4×10^5 eGFP-CD34+. At 60 days post-transplant we performed flow cytometric analysis and Colony Forming Unit assay (CFU) of PB and BM to assess the levels of donor cell engraftment. Confocal microscopic analysis of bone sections was also performed in order to identify the localization and interaction between the transplanted HSC and MSC. Animals receiving mKateMSC 3 days prior to HSC transplantation displayed a 1.6 and a 1.1 fold increase in circulating donor GFP+cells and donor GFP+BM cells, respectively than animals receiving MSC+HSC simultaneously. However the latter had significantly higher levels of CD34 engraftment in BM (4.5-fold) than animals receiving mKateMSC 3 days prior to HSC transplantation. This also corresponded to higher levels of GFP+CFU in animals transplanted with MSC+HSC simultaneously. Confocal microscopy revealed that regardless of whether animals received mKateMSC 3 days prior to HSC or MSC+HSC simultaneously, HSC and MSC engrafted in clusters; however, there was no preferential interaction of the transplanted HSC with autologous MSC over the recipient's own cells. Nevertheless, animals receiving mKateMSC 3 days prior to HSC had higher levels of MSC in their BM than animals receiving MSC+HSC simultaneously. Furthermore, independent of the regimen of cells transplanted, depending on the site of bone engraftment, i.e., metaphysis or diaphysis, transplanted HSC localized preferentially in perivascular areas in the diaphysis, while in the metaphysic they appeared to contribute to the osteoblastic cell layer coating the ossifying bone. Also, HSC contributed to the osteoblastic layer more consistently and robustly than the transplanted MSC. These results show that the delivery of MSC prior to HSC results in higher levels of MSC engraftment in the bone marrow and higher levels of donor derived blood cells in circulation. However, the presence of MSC in the transplanted graft is necessary for optimal engraftment of CD34+ cells. Furthermore, CD34+ cells, and not MSC, migrated efficiently to the metaphysis where they were able to integrate into the developing bone and contribute to the osteoblastic layer. Disclosures: No relevant conflicts of interest to declare.

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Optimization Of Vascular Niches To Increase Hematopoietic Engraftment

Blood ◽

10.1182/blood.v122.21.4456.4456 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4456-4456 ◽

Cited By ~ 3

Author(s):

Saloomeh Mokhtari ◽

Evan Colletti ◽

Christopher D Porada ◽

Graca Almeida-Porada

Keyword(s):

Bone Marrow ◽

Lentiviral Vector ◽

In Utero ◽

Type I ◽

Cellular Interactions ◽

Inherited Disorders ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Cd34 Cells ◽

Hsc Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) is a promising approach for correcting selected congenital hematologic and immunologic disorders. However, if higher levels of donor hematopoietic stem/progenitor (HSC) cell engraftment could be achieved, a wider range of inherited disorders could be targeted. We have previously shown that adult bone marrow (BM) derived-CD34+ cells adhere less efficiently to fetal stromal cells than to their adult counterpart. Furthermore, it has been shown that perivascular cells are able to support, through cellular interactions, the long-term engrafting HSC. Here, we hypothesized that by transplanting bone marrow (BM)-derived endothelial progenitor cells (EPC) prior to HSC transplantation, it would be possible to establish HSC donor-optimized vascular niches within the recipient’s BM, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Adult sheep BM HSC were immunoselected with an antibody against sheep CD34, while EPC were isolated by adherence to collagen type I. Characterization of these cells demonstrated that they were spindle-shaped, and they expressed fetal liver kinase (flk-1/KDR), vonWillebrand factor (vWF), and melanoma cell adhesion molecule (MCAM/CD146). In addition, these cells formed capillary-like structures in Matrigel-based media. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered, intraperitoneally, 1.4X105 CD34+ cells transduced with an eGFP-encoding lentiviral vector (HSCeGFP) in combination with 7.1X105 EPC transduced with an mKate-encoding lentiviral vector (EPCmKate) (n=4), from the same donor, either concurrently, or 3 days prior to HSCeGFP transplantation. At 60 days post-transplant, we performed flow cytometry on peripheral blood (PB) and BM to assess the levels of donor cell engraftment. We also performed confocal microscopic analysis of bone sections to identify the localization and interaction between transplanted cells. Our results demonstrate that animals receiving EPCmKate 3 days prior to HSC transplantation displayed 13-fold higher levels of eGFP(+) hematopoietic cells in their BM (6.5±0.5%), when compared with animals receiving EPC and HSC simultaneously (0.39±0.29%). Confocal microscopy analysis showed that, regardless of the time-point of transplant, donor cells that engrafted in the diaphysis localized to the perivascular area, and a correlation was found between the levels of CD146(+)mKate cells and HSCeGFP engraftment. By contrast, in the metaphysis, only eGFP(+) cells were detected, and these cells co-expressed osteopontin, a marker of osteoblasts. These results show that in IUHSCT, delivery of EPC,CD146(+), cells prior to CD34+HSC results in modification of the vascular niches by donor-derived cells, leading to significantly higher levels of HSC engraftment. Furthermore, a considerable percentage of CD34+eGFP(+) cells did not contribute to the hematopoietic pool, but rather, contributed to the developing bone, suggesting that a more effective selective process for HSC might be necessary for improving engraftment in IUHSCT. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽

10.1182/blood.v112.11.821.821 ◽

2008 ◽

Vol 112 (11) ◽

pp. 821-821 ◽

Cited By ~ 3

Author(s):

Marina Cavazzana-Calvo ◽

Nathalie Cartier ◽

Salima Hacein-Bey Abina ◽

Gabor Veres ◽

Manfred Schmidt ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Stem Cell ◽

Lentiviral Vector ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Post Transplant ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

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A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽

10.1182/blood.v114.22.692.692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 692-692

Author(s):

Naoya Uchida ◽

Phillip W Hargrove ◽

Kareem Washington ◽

Coen J. Lap ◽

Matthew M. Hsieh ◽

...

Keyword(s):

T Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Autologous Transplantation ◽

Vector System ◽

Transduction Efficiency ◽

Large Animal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

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Hyaluronan Expressed by Bone Marrow Mesenchymal Cells Regulates Functions of the Hematopoietic Microenvironment

Blood ◽

10.1182/blood.v120.21.1243.1243 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1243-1243

Author(s):

Valentina Goncharova ◽

Ingrid U Schraufstaetter ◽

Shinji Iizuka ◽

Yu Yamaguchi ◽

Sophia K Khaldoyanidi

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Biologically Active ◽

Dimensional Structure ◽

Optimal Time ◽

Hematopoietic Stem ◽

Hematopoietic Microenvironment ◽

Hematopoietic Recovery ◽

Hsc Transplantation

Abstract Abstract 1243 While the quantity and quality of transplanted hematopoietic stem cells (HSC) are important for the recovery of hematopoiesis, the functional status of the regulatory hematopoietic microenvironment is a critical parameter that determines the regenerative function of HSCs. The quality of the microenvironment, i.e. its ability to support hematopoiesis, may be compromised under pathological circumstances such as during disease development or as a result of therapeutic interventions. Thus, the hematopoietic microenvironment should be allowed to recover prior to HSC transplantation. To effectively prepare the marrow microenvironment for HSC transplantation it is important to understand which of the molecular pathways regulating the function of the microenvironment are disrupted under the specific pathological condition. The involvement of hyaluronan (HA) in regulation of hematopoiesis has been previously suggested. However, whether HA contributes to the regulatory network of the hematopoietic microenvironment is not well understood. Since HA is highly susceptible to irradiation, which induces HA degradation and depolymerization leading to HA chain fragmentation and affecting its three-dimensional structure, sublethally irradiated mice (6Gy) were used to test the effect of exogenous HA on hematopoietic recovery. We found that administration of HA shortened the period of cytopenia compared to control mice which received vehicle only. To investigate whether the depletion of HA from the microenvironment has negative effects of hematopoietic homeostasis, knockout mice of three hyaluronan synthase genes (Has1, Has2, Has3) were generated as a mouse model of targeted HA deficiency in the hematopoietic microenvironment. Specifically, we generated double Has knockout (KO) mice (dHAS1/3 KO, Has1–/–;Has3–/–) and triple Has KO mice (tHAS1/2/3 KO, Prx1-Cre;Has2flox/flox;Has1–/–;Has3–/–). In the following study, wild type (WT), dHAS1/3 and tHAS1/2/3 KO mice were sublethally irradiated (6Gy) and the dynamics of hematopoietic recovery were tested. We found that the recovery of leukocytes in tHAS1/2/3 KO mice was significantly delayed as compared to WT and dHAS1/3 KO mice. This finding suggests that the HA-deficient microenvironment cannot support hematopoietic recovery following irradiation. Additional tests demonstrated that the number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of tHAS1/2/3 KO mice as compared to WT and dHAS1/3/KO mice. In line with this observation, decreased hematopoietic activity was observed in long-term bone marrow cultures (LTBMC) from tHAS1/2/3 KO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dHAS1/3/KO cultures was the same. 4-methylumbelliferone (4-MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3 and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of HSC in response to SDF-1. In addition, pharmacological inhibition of HA synthesis decreased homing of transplanted HSC into the marrow and interactions of HSC with endothelial cells under conditional physiological shear stress. Our findings demonstrate that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment. Collectively, our results strongly suggest that HA is a biologically active component of the hematopoietic microenvironment and is involved in regulating hematopoietic homeostasis. Since some treatments or compounds reduce HA concentrations in tissues and some conditions are associated with increased levels of HA, it may prove clinically useful to monitor the dynamics of endogenous HA recovery to aid in identifying the optimal time for stem cell transplantation. Our data also suggest that biologically active exogenous HA polymers of the correct size, source, and conformation as well as HA synthesis inhibitors may have potential use in clinical hematology to correct misbalanced HA levels. Disclosures: No relevant conflicts of interest to declare.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽

10.1182/blood.v122.21.1192.1192 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1192-1192

Author(s):

Hirotaka Kawano ◽

Tomotoshi Marumoto ◽

Takafumi Hiramoto ◽

Michiyo Okada ◽

Tomoko Inoue ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Embryonic Stem ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

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Vitamin K2 Supports Hematopoiesis through Acting on Bone Marrow Mesenchymal Stromal/Stem Cells

Blood ◽

10.1182/blood.v126.23.1192.1192 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1192-1192 ◽

Cited By ~ 2

Author(s):

Aya Fujishiro ◽

Yasuo Miura ◽

Masaki Iwasa ◽

Sumie Fujii ◽

Akihiro Tamura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometric Analysis ◽

Hematopoietic Cells ◽

Vitamin K2 ◽

Therapeutic Effects ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells

Abstract [Background] Myelodysplastic syndrome is an intractable disorder characterized by ineffective hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is the only curative therapy for eligible patients, hematopoiesis-supportive pharmacotherapy is practically important for transplant-ineligible patients to overcome transfusion dependency and infections. Vitamin K2 (VK2, menatetrenone) is a drug used to aim at improvement of hematopoiesis in MDS patients (Leukemia 14: 1156, 2000). However, the exact mechanism how VK2 improves hematopoiesis remains largely unknown. It was reported that VK2 induces MDS cells to undergo apoptosis (Leukemia 13: 1399, 1999). Here, we investigated our hypothesis that VK2 exerts its hematopoiesis-supportive effects through acting on mesenchymal stem/stromal cells (BM-MSCs) in the bone marrow microenvironment. [Methods] Normal bone marrow (BM) samples from healthy adult volunteers were purchased from AllCells (Emeryville, CA). BM-CD34+ cells were isolated from BM-mononuclear cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human BM-MSCs were isolated according to our previously published methods (Stem Cells 32:2245, 2014). In co-culture experiments, BM-MSCs with or without VK2 treatment were seeded on a 24-well culture plate. BM-CD34+ cells were applied on the MSC-grown plate and co-cultured in SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, 50 ng/mL TPO and 20 ng/mL IL-3. After 10 days of co-culture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis. [Results] We first tested the direct effect of VK2 on BM-CD34+ cells. BM-CD34+ cells were treated with VK2 at various concentrations ranged from 0 µM to 10 µM for 24 hours and then cultured in SFEM in combinations with cytokines. Surprisingly, viable hematopoietic cells were hardly detected in the expansion culture of BM-CD34+ cells treated with 10 µM VK2. Even with 1 µM treatment, the number of CD45+ cells was decreased, as compared to that of expansion culture of untreated BM-CD34+ cells. The apoptosis analysis showed that the percentage of AnnexinV+ PI+ cells in the expanded hematopoietic cells is increased by VK2 treatment. We next examined the effect of VK2 on the hematopoiesis-supportive capability of BM-MSCs. BM-MSCs were pretreated with VK2 at various concentrations and then co-cultured with BM-CD34+ cells. The numbers of CD34+ cells and CD45+ cells were increased in a VK2 dose-dependent manner. These results demonstrated that VK2 shows different effects on distinct stem/progenitor cells: the induction of apoptosis in BM-CD34+ cells and the enhancement of hematopoiesis-supportive capability of BM-MSCs. We then investigated whether apoptosis-related cell death of BM-CD34+ cells by VK2 treatment is ameliorated in the presence of BM-MSCs. Both BM-CD34+ cells and BM-MSCs were treated with VK2 for 24 hours, and then co-cultured. The number of CD34+ cells was not decreased significantly in contrast to its severe decrease in single culture of VK2-treated BM-CD34+ cells. We further analyzed the effect of VK2 on BM-MSCs. Subpopulation analysis in co-culture of CD34+ cells with VK2-treated BM-MSCs showed that the expansion efficacy of CD34+CD38+ cells is higher in comparison to that of CD34+CD38- cells. In addition, the percentages of CD34-CD33+ cells and CD34-CD13+ cells were higher than those in co-cultures with untreated BM-MSCs. Therefore, VK2-treated BM-MSCs supported the expanded CD34+ cells to skew their phenotype toward myeloid lineage. The presence of a transwell in the co-culture system was unrelated to the expansion pattern of CD34+ cells, which suggested the involvement of soluble factors with respect to the underlining mechanism. We therefore compared the levels of hematopoiesis-supporting cytokine mRNA expression in VK2-treated and untreated BM-MSCs: VK2-treated BM-MSCs showed lower expression of CXCL12/SDF-1 mRNA and a trend toward higher expression of GM-CSF mRNA. [Summary] VK2 acted on BM-MSCs to support their ability to enhance expansion and myeloid differentiation of BM-CD34+ cells probably via altered GM-CSF and CXCL12/SDF-1 expression in MSCs. These findings may help to identify the mechanisms of therapeutic effects of VK2 in patients with MDS (Figure). Disclosures No relevant conflicts of interest to declare.

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Inhibition of Lysine-Specific Demethylase 1A (LSD1) Supports Human HSCs in Culture By Preserving Their Immature State

Blood ◽

10.1182/blood-2018-194 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 194-194

Author(s):

Agatheeswaran Subramaniam ◽

Mehrnaz Safaee Talkhoncheh ◽

Kristijonas Zemaitis ◽

Shubhranshu Debnath ◽

Jun Chen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stem Cell ◽

Cell Division ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Enrichment Score ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract The molecular mechanisms that govern hematopoietic stem cell (HSC) fate decisions remain incompletely defined. It has been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. Lysine-specific histone demethylase 1A (LSD1), also known as lysine (K)-specific demethylase 1A (KDM1A), regulates gene expression by specifically eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues. Studies in mice have shown that, conditional knockdown of LSD1 results in an expansion of bone marrow hematopoietic stem and progenitor cells (HSPCs). However, a complete knockout of LSD1 results in pancytopenia and a dramatic reduction of HSPCs. In this study, we asked whether inhibition of LSD1 would improve the maintenance or expansion of cultured human HSCs derived from umbilical cord blood (UCB). To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) at their respective IC50 values (20µM, 16nM and 70nM) and expanded the cultures for 6 days in serum free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Since we (Subramaniam et. al. Haematologica 2018) and others recently have shown that EPCR is a reliable cell surface marker to track UCB derived HSCs during in vitro culture, we quantified the numbers of CD34+EPCR+ cells using flow cytometry and compared to DMSO treated control cultures. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of CD34+EPCR+ cells, compared to controls. Further, from dose response experiments we found that 2-PCPA at 1.25 µM expanded the total CD34+ cell population more efficiently than GSK-LSD1, and we therefore used 2-PCPA at this concentration for the subsequent experiments. Using carboxyfluorescein succinimidyl ester (CFSE) labeling to monitor cell division, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells compared to DMSO controls, suggesting that the expansion of CD34+EPCR+ cells is not due to increased proliferation, and that LSD1 inhibition rather may prevent differentiation of the immature HSPCs. To further explore this, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs using gene expression profiling of CD34+CD38-CD45RA-CD90+ cells following 24 hours of culture with or without 2-PCPA treatment. We found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment compared to DMSO control (Normalized Enrichment Score (NES)=1.49, q=0.05). This suggest that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Strikingly, the gene signature induced by LSD1 inhibition was highly similar to that induced by the known HSC expanding compound UM171 (NES=1.43, q=0.11). UM171 is a molecule with unknown target and has also been shown to dramatically expand the EPCR+ population in culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). LDA was performed by transplanting 4 doses (day 0 equivalents of 20000, 1000, 300 and 100 CD34+ cells) of DMSO and 2-PCPA treated cultures into sub lethally irradiated (300cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human CD45+ cell engraftment in the bone marrow was analyzed 18 weeks' post transplantation. Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells per day 0 CD34+ cell equivalent compared to the DMSO control (1 in 615 vs 1 in 3041, p=0.03). Thus, the 2-PCPA treated cultures had significantly enhanced HSCs numbers. To determine the absolute expansion rate, we are currently performing LDA using uncultured cells as well. Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state. Currently we are exploring the possibilities of using LSD1 inhibitors in combination with other known modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽

10.1182/blood.v124.21.4358.4358 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4358-4358

Author(s):

Manal Alsheikh ◽

Roya Pasha ◽

Nicolas Pineault

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Soluble Factors ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Impact ◽

Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

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Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.4810.4810 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4810-4810

Author(s):

Olga Kulemina ◽

Izida Minullina ◽

Sergey Anisimov ◽

Renata Dmitrieva ◽

Andrey Zaritskey

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Feeder Layers

Abstract Abstract 4810 Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation. In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression. Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression. Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01).Figure 1.Cord Blood HSPC ex vivo expansionFigure 1. Cord Blood HSPC ex vivo expansionFigure 2.Bone Marrow HSPC ex vivo expansionFigure 2. Bone Marrow HSPC ex vivo expansion Conclusion: BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures: No relevant conflicts of interest to declare.

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