Optimization Of Vascular Niches To Increase Hematopoietic Engraftment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4456-4456 ◽  
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.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 940-940
Author(s):  
Evan Colletti ◽  
Saloomeh Mokhtari ◽  
Chad Sanada ◽  
Esmail D Zanjani ◽  
Christopher D Porada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Flow Cytometric Analysis ◽  
Microscopic Analysis ◽  
Donor Cell ◽  
Osteoblastic Cell ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

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.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
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)

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 ◽  
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.

Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3620-3627 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Nam Tran ◽  
Esmail D. Zanjani
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
In Utero ◽  
Human Cells ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Hsc Transplantation

Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

Heme-Related Blood Disorders

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-18-SCI-18
Author(s):  
Hervé Puy ◽  
Karim Zoubida ◽  
Lyoumi Said ◽  
Lydie M. Da Costa ◽  
Gouya Laurent
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Modifier Gene ◽  
Heme Biosynthesis ◽  
Type I ◽  
Erythroid Cells ◽  
Inherited Disorders ◽  
Sideroblastic Anemia ◽  
Hematopoietic Stem ◽  
In Erythroid Cells

Abstract Heme biosynthesis in erythroid cells is intended primarily for the formation of hemoglobin. As in every cell, this synthesis requires a multi-step pathway that involves eight enzymes including the erythroid-specific δ-aminolevulinate synthase (ALAS2, the first regulated enzyme that converts glycine and succinyl CoA into ALA) and the ubiquitous ferrochelatase (FECH, the final enzyme). Heme biosynthesis also requires membrane transporters that are necessary to translocate glycine, precursors of heme, and heme itself between the mitochondria and the cytosol. Defects in normal porphyrin and/or heme synthesis and transport cause four major erythroid inherited disorders, which may or may not be associated with dyserythropoiesis (e.g., sideroblastic, microcytic anemia and/or hemolytic anemia): "X-linked" sideroblastic anemia (XLSA) and X-linked dominant protoporphyria (XLDPP) are two different and opposing disorders but related to altered gene encoding ALAS2 only. Defective activity of this enzyme due to mutations in the ALAS2 gene causes the XLSA phenotype, including microcytic, hypochromic anemia with abundant ringed sideroblasts in the bone marrow. Vice versa, gain-of-function mutations of ALAS2 are responsible of the XLDPP characterized by predominant accumulation of the hydrophobic protoporphyrin (PPIX, the last heme precursor) in the erythrocytes without anemia or sideroblasts. Furthermore, the glycine transporter (SLC25A38) and Glutaredoxin 5 genes are reported to be involved in human non-syndromic sideroblastic anemia. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. CEP leads to excessive synthesis and accumulation of type I isomers of porphyrins in the reticulocytes, followed by intravascular hemolysis and severe anemia. The ALAS2 gene may act as a modifier gene in CEP patients (Figueras J et al, Blood. 2011;118(6):1443-51). Erythropoietic protoporphyria (EPP) results from a partial deficiency of FECH and leads similarly to XLDPP, to deleterious accumulation of PPIX in erythroid cells. Most EPP patients present intrans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. Abnormal spliced mRNA is degraded which contributes to the lowest FECH enzyme activity and allowed EPP phenotype expression. We have identified an antisense oligonucleotide (ASO) to redirect splicing from cryptic to physiological site and showed that the ASO-based therapy mediates normal splice rescue of FECH mRNA and reduction by 60 percent of PPIX overproduction in primary cultures of EPP erythroid progenitors. Therapeutic approaches to target both ALAS2 inhibition and heme-level reduction may be useful in other erythroid disorders such as thalassemia (where reduced heme biosynthesis was shown to improve the clinical phenotype) or the Diamond-Blackfan anemia (DBA). Indeed, in some DBA patients, an unusual mRNA splicing of heme exporter FLVCR has been found, reminiscent of Flvcr1-deficient mice that develop a DBA-like phenotype with erythroid heme accumulation. Thus, FLVCR may act as a modifier gene for DBA phenotypic variability. Recent advances in understanding the pathogenesis and molecular genetic heterogeneity of heme-related disorders have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias and some porphyrins and heme transporters, new understanding of the pathogenesis of the erythropoietic disorders, and new and experimental treatments such as chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for erythropoietic porphyrias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adult bone marrow-derived pluripotent hematopoietic stem cells are engraftable when transferred in utero into moderately anemic fetal recipients

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 833-841 ◽  
Author(s):  
BR Blazar ◽  
PA Taylor ◽  
DA Vallera
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
In Utero ◽  
Donor Strain ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Cell Engraftment ◽  
Adult Bone

We have used W41/W41 (C57BL/6-Ly 5.1, Gpi-1b) anemic mice and a newly developed double congenic donor strain (C57BL/6-Ly 5.2, Gpi-1a) to determine if adult bone marrow (BM) injected in utero could provide stem cell engraftment. Of 38 fetuses injected intraperitoneally on day 13/14 of gestation with donor BM cells, 17 (47%) were live-born. On day 6, 12% had erythroid engraftment. On day 59, in 50% (8/16) of mice, 50% to 75% of erythroid cells, 42% of T cells, 5% of B cells, and 26% of granulocytes in the peripheral blood (PB) were derived from the in utero-injected donor BM. At 141 days, thymic, splenic, lymph node, BM, and PB chimerism studies showed that 57% to 80% of T cells, 10% to 15% of B cells, and 27% to 43% of granulocytes were of donor origin. At this time, BM was injected into irradiated secondary recipients. On day 104 posttransfer, a mean 23% of T cells, 8% of B cells, and 40% of granulocytes were derived from the in utero donor BM. These data indicate that adult BM has hierarchical engraftment capabilities in W41/W41 mice and prove that stem cells are engraftable in utero.

An in-Vivo Model of T Cell-Mediated Rejection of Human Hematopoietic CD34+ Stem Cells Using NOD/SCID γnull (NOG) Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4474-4474
Author(s):  
Benedetta Nicolini ◽  
Dolores Mahmud ◽  
Nadim Mahmud ◽  
Giuseppina Nucifora ◽  
Damiano Rondelli
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Mouse Model ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Abstract 4474 We have previously demonstrated that human CD34+ cells include subsets of antigen presenting cells capable of stimulating anti-stem cell T cell alloreactivity in-vitro. In this study we transplanted human CD34+ cells and allogeneic T cells in a NOD/SCID γnull (NOG) mouse model and evaluated the occurrence of stem cell rejection as well as xenogeneic graft-versus-host disease (GVHD) following the infusion of different doses of T cells. After sublethal irradiation NOG mice were cotransplanted with 2×105 CD34+ cells and HLA mismatched CD4+CD25- T cells at 1:0 (control), 1:2 or 1:10 CD34+ cell: T cell ratio (n=5-10 mice per group). Hematopoietic stem cell and T cell engraftment was assessed in the bone marrow and in the spleen 6 weeks following transplantation or earlier in case the animals died. Control mice transplanted with CD34+ cells alone showed a high level of stem cell engraftment (huCD45+ cells: 60±10%) in the bone marrow, encompassing CD19+ B cells (64±4%), CD34+ cells (18±1%), CD33+ myeloid cells (7±1%), CD14+ monocytes (3±1%), and no T cells within huCD45+ cells. In contrast, mice that were transplanted with CD34+ cells and 4×105 (1:2 ratio) or 2×106 (1:10 ratio) T cells had only 9±2% and 3±1% huCD45+ cells, respectively, in the bone marrow (p=0.01). Moreover, marrow samples of mice cotransplanted with CD34+ cells and T cells at 1:2 or 1:10 ratio included >98% huCD3+ T cells and no CD34+ cells. Spleen engraftment of huCD45+ cells was lower (25±8%) in control mice (1:0 ratio) as compared to 66±10% and 36±11% in 1:2 and 1:10 groups, respectively (p=0.05). As observed in the marrow, also the spleen of animals receiving CD34+ and T cells included >98% CD3+ T cells. Among the T cells, both in the marrow and in the spleen of mice in the 1:2 and 1:10 ratio groups, 60-70% were CD4+CD8- cells, 22-25% CD8+CD4- cells, 1-3% CD56+ cells, and 2-5% CD4+CD25+ cells. In mice receiving 4 ×105 T cells (1:2 ratio), on average 12±6% of the T cells in the bone marrow and spleen were CD4+CD8+. Only mice receiving 2×106 T cells (1:10 ratio) showed GVHD. This was demonstrated by fur changes, reduced survival (p=0.02) and weight loss (p=0.0001) compared to control mice or mice receiving a lower dose of T cells (1:2 ratio). The marrow engraftment of CD3+ cells with disappearance of CD34+ cells in mice receiving low doses of allogeneic T cells, in the absence of evident xenogeneic GVHD, suggests that NOG mouse model represents a useful tool to study human stem cell rejection. This model will be also utilized to investigate new strategies of immunosuppressive cell therapy applied to stem cell transplantation in an HLA mismatched setting. Disclosures: No relevant conflicts of interest to declare.

Enhanced Engraftment and Maintenance of CXCR4 Expressing CD34+ Cells In Vivo Is Related to Microenvironmental Interactions, Rather Than Direct Receptor Signaling to Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2306-2306 ◽  
Author(s):  
Naomi J. Anderson ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cell ◽  
Hematopoietic Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Interaction of the chemokine receptor CXCR4 with its ligand SDF-1α (SDF) has been reported to play an important role in engraftment of hematopoietic stem cells (HSC) in the bone marrow (BM). However a critical requirement for CXCR4 in HSC engraftment is still controversial. It also remains unclear whether the effects that CXCR4 has on hematopoietic cell engraftment are related to enhanced homing of HSC to the bone marrow cavity, increased retention in marrow microenvironment or direct and indirect effects of CXCR4 stimulation on stem and progenitor cell proliferation, self-renewal and survival. To address these questions we have overexpressed CXCR4 in human cord blood CD34+ cells by transduction with an MSCV retroviral vector containing CXCR4 and eGFP (MIG-CXCR4). CXCR4 overexpressing cells were compared with control cells transduced with vectors expressing eGFP alone (MIG). CD34+eGFP+ cells were selected after transduction by flow cytometry sorting. We confirmed that CD34+ cells transduced with the MIG-CXCR4 vector demonstrated increased CXCR4 expression compared with MIG vector transduced controls (mean channel fluorescence for CXCR4 was 340±77.8 for MIG-CXCR4 transduced CD34+ cells compared with 142±37.1 for MIG transduced cells, n=8). MIG-CXCR4 transduced CD34+ cells demonstrated significantly enhanced chemotaxis to SDF in transwell migration assays (36±2% migration for MIG-CXCR4 vs. 20±4% migration for MIG transduced CD34+ cells to 100nM SDF-1, n=4, p=0.05). CD34+ cells transduced with MIG-CXCR4 demonstrated a 1.52±0.4 fold increase in expansion of total cell number compared with controls after 1 week of in vitro culture with growth factors (GF) [SCF (50ng/ml), TPO (100ng/ml), FL (100ng/ml), SDF (60ng/ml), n=3]. However, enhanced cellular expansion was not sustained on further GF culture. To evaluate the effect of CXCR4 overexpression on in vivo engraftment, CD34+ cells transduced with MIG-CXCR4 and MIG vectors were injected intravenously into sublethally irradiated NOD/SCID mice and human hematopoietic cell engraftment was evaluated after 6–8 weeks. MIG-CXCR4 transduced cells demonstrated significantly higher levels of engraftment with human CD45+ cells compared with MIG transduced cells (8±4.8% vs. 0.22±0.07% CD45+ cells in bone marrow, 1.3±0.9% vs. 0.2±0.09% CD45+ cells in spleen, and 1.8±1.0% vs. 0.3±0.25% CD45+ cells in peripheral blood for MIG-CXCR4 vs. MIG transduced cells, respectively, n=5). In addition, markedly higher levels of CD34+ cell engraftment was observed in the bone marrow of animals receiving MIG-CXCR4 vs. MIG transduced cells (1.7±1.0% vs. 0.06±0.03% CD34+ cells respectively, n=5). Consistent with this, the human CFC frequency in bone marrow of mice receiving MIG-CXCR4 transduced CD34+ cells was increased compared to mice receiving MIG transduced cells (31±0.5 CFC/100,000 cells vs. 5±3.2 CFC/100,000 cells, n=2,3, respectively). In conclusion, our results indicate that ectopic expression of CXCR4 in CD34+ cells results in enhanced engraftment of human hematopoietic cells and increased maintenance of hematopoietic stem and progenitor cells in the NOD/SCID mouse model. The effects of CXCR4 overexpression are considerably more prominent in vivo than in direct in vitro assays. It therefore appears that altered stem and progenitor cell homing and microenvironmental interaction, rather than direct signaling to HSC, may be responsible for enhanced CD34+ cell engraftment and maintenance following CXCR4 receptor overexpression.

Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3620-3627 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Nam Tran ◽  
Esmail D. Zanjani
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
In Utero ◽  
Human Cells ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Hsc Transplantation

Abstract Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3478-3482 ◽  
Author(s):  
Xiuli Wang ◽  
Michael Rosol ◽  
Shundi Ge ◽  
Denise Peterson ◽  
George McNamara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Signal Intensity ◽  
Bioluminescence Imaging ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
In Vivo Bioluminescence Imaging

Abstract The standard approach to assess hematopoietic stem cell (HSC) engraftment in experimental bone marrow transplantation models relies on detection of donor hematopoietic cells in host bone marrow following death; this approach provides data from only a single time point after transplantation for each animal. In vivo bioluminescence imaging was therefore explored as a method to gain a dynamic, longitudinal profile of human HSC engraftment in a living xenogeneic model. Luciferase expression using a lentiviral vector allowed detection of distinctly different patterns of engraftment kinetics from human CD34+ and CD34+CD38- populations in the marrow NOD/SCID/β2mnull mice. Imaging showed an early peak (day 13) of engraftment from CD34+ cells followed by a rapid decline in signal. Engraftment from the more primitive CD34+CD38- population was relatively delayed but by day 36 increased to significantly higher levels than those from CD34+ cells (P < .05). Signal intensity from CD34+CD38--engrafted mice continued to increase during more than 100 days of analysis. Flow cytometry analysis of bone marrow from mice after death demonstrated that levels of 1% donor cell engraftment could be readily detected by bioluminescence imaging; higher engraftment levels corresponded to higher image signal intensity. In vivo bioluminescence imaging provides a novel method to track the dynamics of engraftment of human HSC and progenitors in vivo. (Blood. 2003;102: 3478-3482)

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