In Vivo Expansion of Megakaryocyte Progenitors in CD26−/ − Mice: Implications for Novel Post-Transplant Therapeutics.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1188-1188
Author(s):  
Shannon Kidd ◽  
Carlos E. Bueso-Ramos ◽  
Laura A. Paganessi ◽  
Henry Fung ◽  
Stephanie Gregory ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Severe Thrombocytopenia ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Post Transplant ◽  
Complete Blood Counts ◽  
And Function

Abstract Hematopoietic stem cell transplantation (HSCT) is a successful treatment option for patients with malignant or non-malignant severe hematologic diseases. Readily available umbilical cord blood (CB) has emerged as an important donor source that has a lower histocompatability requirement and carries a reduced risk of graft vs. host disease. However, a frequent complication of CB use is delayed hematopoietic recovery, in particular long lasting severe thrombocytopenia (median of 117 days to platelet recovery, COBLT 2005). To overcome this clinically, effective strategies for enhancing megakaryopoiesis, thrombopoiesis, or both are needed. We have previously described the importance of CD26 (dipeptidylpeptidase IV) in engraftment (Christopherson, KW 2nd, et al, Science2004. 305:1000–3). However, the involvement of CD26 in megakaryopoiesis has not been investigated. We hypothesized that CD26 acts to suppress megakaryopoiesis and that removal of CD26 activity would result in expansion of the megakaryocyte progenitor population in vivo. To test this hypothesis, we evaluated megakaryocyte (MK) development in the context of development of other mature blood cells in CD26 deficient (CD26−/ −) mice as compared to control C57BL/6 mice. Histological analysis of formalin fixed paraffin embedded tissue sections and peripheral blood cytopsins revealed an increased presence of MK in the bone marrow, spleen, thymus and peripheral blood. However, complete blood counts (CBC) suggest no difference in white blood cell, neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet counts. Flow cytometric analysis also revealed no significant changes in CD3, CD4, or CD8 T-cells; B220 B-cells; and Gr-1/Mac-1 granulocytes/neutrophils in the bone marrow, spleen, thymus, or peripheral blood as appropriate. There was also no change in the percentage of peripheral blood CD41 MK but there was a increase from 0.09% in C57BL/6 BM to 0.26% CD41+Sca-1+c-kit− MK progenitors in the CD26−/ − BM (P≤0.05). Methylcellulose based myeloid progenitor assays did also reveal respective CFU-GM, BFU-E, and CFU-GEMM per femur values (mean±SEM) of 24480±1426, 1448±154.86, and 852±41 for C57BL/6 BM cells; 24451±1342, 974.29±81, and 1634±177 for CD26−/ − BM cells. This represents a 92% increase in CFU-GEMM and corresponding 30% decrease in BFU-E in the CD26−/ − mouse BM (P≤0.01, n=7 mice/group). Collagen based megakaryocyte progenitor assays (CFU-MK) revealed a 25% increase from 4455.00±207.62 colonies/femur in C57BL/6 BM to 5555.00±608.12 in CD26−/ − BM (P≤0.05, n=3 mice/group). These results establish a basis on which to propose that CD26 may act to regulate early events in megakaryocyte progenitor formation and function. They also suggest that the use of CD26 inhibitors may have a beneficial effect on improved megakaryocyte progenitor function and/or reconstitution post-transplant.

Nfi Genes Are Novel Regulators Of Murine Hematopoietic Stem- and Progenitor Cell Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 735-735
Author(s):  
Per Holmfeldt ◽  
Pardieck Jennifer ◽  
Shannon McKinney-Freeman
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Family ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Hematopoietic stem cells (HSCs) are responsible for life-long maintenance of hematopoiesis. HSC transplantation represents one of the most heavily exploited cell based therapies, routinely used to treat a myriad of life threating disorders, such as leukemia and bone marrow failure. Identifying the molecular pathways that regulate HSC engraftment is crucial to further improving outcomes in patients that rely on HSC transplantation as a curative therapy. By examining the global gene expression profiles of highly purified HSC (Lineage-Sca-1+c-Kit+CD150+CD48-), we recently identified the following members of the Nfi gene family of transcription factors as highly expressed by HSC (McKinney-Freeman et al., Cell Stem Cell, 2012): Nfix, Nfia, and Nfic. These data suggest that Nfi genes may play a novel role in regulating HSC function. To test this hypothesis, HSCs were enriched from adult bone marrow (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced, individually, with lentiviruses carrying shRNAs targeting each Nfi gene. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced bone marrow LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfi-depleted cells. Although shRNA mediated knockdown of Nfi gene expression had no effect on the in vitro cell growth or viability of LSK cells, Nfi-depleted HSCs displayed a significant loss of short- and long-term in vivo hematopoietic repopulating activity. This was true for Nfia-, Nfic-, and Nfix-deficient HSC. While Nfia and Nfic are only expressed by bone marrow HSC, Nfix is highly expressed by both bone marrow and fetal liver HSC. When Nfix was depleted by shRNAs from LSK cells purified from E14.5 fetal liver, a similar loss in competitive repopulating potential was seen. Lineage analysis of peripheral blood of recipients showed no significant differences in the distribution of the major blood lineages derived from LSK cells transduced with Nfi-specific shRNAs compared to controls. When the bone marrow of recipients transplanted with Nfix- depleted cells was examined 4 and 16 weeks post-transplant, a general loss of all hematopoietic stem- and progenitor compartments examined was seen relative to control. Thus, the observed decrease in repopulating activity occurs at the level of HSCs and multipotent progenitors. To confirm an essential role for an Nfi gene family member in the regulation of HSC engraftment post-transplant, LSK cells were purified from Nfix fl/fl mice, transduced with lentiviral Cre recombinase and subsequently introduced into lethally irradiated recipients alongside congenic competitor cells. Like LSK transduced with Nfix-specific shRNAs, Nfix-/- LSK cells failed to repopulate the peripheral blood of recipient mice as efficiently as control and similar trends were detected in all stem- and progenitor cell populations examined. Time-course experiments immediately following transplantation revealed that Nfix-depleted LSK cells establish themselves in the marrow of recipient mice as efficiently as control at 5 days post-transplant, but thereafter exhausted rapidly. Examination 10 days post-transplant revealed a 5-fold increase in apoptosis specifically in the LSK compartment, but not in its differentiated progeny, in recipients transplanted with Nfix-depleted LSK cells compared to control. The increase in apoptosis was not associated with any apparent change in the cell cycle status of the LSK cells. These data suggest that Nfi genes are necessary for the survival of HSC post-transplantation. In an effort to identify the molecular pathways regulated by Nfi genes in HSC, we acquired the global gene expression profiles of Nfix-depleted HSC. In agreement with our observation that Nfix-deficient HSC displays elevated levels of apoptosis following transplantation in vivo, we observed a significant decrease in multiple genes known to be important for HSC survival, such as Erg, Mecom and Mpl, in Nfix-depleted HSC. In summary, we have for the first time established a role for the Nfi gene family in HSC biology, as evident by a decrease in bone marrow repopulating activity in Nfi-depleted HSCs. By dissecting the precise role of Nfi genes in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical bone marrow transplantations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Absolute Lymphocyte Count (ALC) Less Than 100 Predicts Adverse Transplant Outcomes with Rabbit Thymoglobulin in Patients Undergoing Matched Unrelated Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4527-4527
Author(s):  
Dipenkumar Modi ◽  
Malini Surapaneni ◽  
Seongho Kim ◽  
Lois Ayash ◽  
Asif Alavi ◽  
...  
Keyword(s):  
T Cell ◽  
Relapse Rate ◽  
Peripheral Blood ◽  
Conditioning Regimen ◽  
Advisory Board ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Transplant Outcomes ◽  
Gvhd Prophylaxis

Introduction: Rabbit thymoglobulin, an in-vivo T-cell depleting agent, is widely used as a part of GVHD prophylaxis regimen. Current dosing of thymoglobulin is often weight based and does not consider patient related factors. This results in highly variable exposure of thymoglobulin. Although higher doses (>7mg/kg) of thymoglobulin have shown to reduce the risk of GVHD, it is associated with increased rate of opportunistic infections and disease recurrence. Conversely, lower dose (2.5mg/kg) of thymoglobulin is associated with increased risk of GVHD. Thus, optimum dosing of thymoglobulin remains undefined. We hypothesized that recipient peripheral blood ALC on the first day of thymoglobulin infusion would interact with the dose of thymoglobulin administered and predict post-transplant outcomes. We plan to identify association of thymoglobulin dose with the ALC on the first day of thymoglobulin. Methods: We retrospectively evaluated clinical outcomes of adult patients (pts) who underwent matched unrelated donor AHSCT and received tacrolimus, mycophenolate (cellcept) and thymoglobulin as GVHD prophylaxis. Thymoglobulin was given at a total dose of 4.5mg/kg in divided fashion (0.5mg/kg on day -3, 1.5mg/kg on day -2 and 2.5mg/kg on day -1). The objectives were to determine rate of GVHD, overall survival (OS), relapse-free survival (RFS), relapse rate and non-relapse mortality (NRM) following AHSCT using Cox proportional hazard regression and competing risk models. Results: Between January 2005 and December 2017, 217 pts underwent AHSCT. The most common indications for AHSCT were AML (n=95, 44%), MDS (n=57, 26%), non-Hodgkin's lymphoma (n=23, 11%), and ALL (n=22, 10%). Median age of pts was 60 years (range, 18-79). All pts received peripheral blood stem cells. Ninety-eight pts (45%) received full intensity conditioning regimen and 119 pts (55%) received reduced intensity regimen. The median ALC on the first day of thymoglobulin administration was 200 K/cubic millimeter. The 6-month cumulative incidence rate (CIR) of grade III-IV acute GVHD was 14.8% and the 2-year CIR of chronic extensive GVHD was 35.4%. With a median follow up of 3.82 years for surviving patients, the 2-year RFS, OS, relapse and NRM were 50%, 57.1, 20.1%, and 30.2%, respectively. CMV and EBV reactivation rates were 37% and 11%, respectively. Four pts developed CMV disease. By our lowest ALC cutoff of 100 K/cubic millimeter, pts were divided into two groups (ALC ≤ 100 vs. ALC > 100). Multivariable analysis revealed that ALC > 100 was associated with significantly superior OS (HR 0.51, 95% CI 0.33-0.79, p=0.002), RFS (HR 0.49, 95% CI 0.33-0.74, p=0.001) and lower NRM (SHR 0.57, 95% CI 0.34-0.97, p=0.038) and marginally lower relapse rate (SHR 0.57, 95% CI 0.31-1.05, p=0.070). In addition, higher infused total nucleated cells was associated with higher NRM (SHR 1.70, 95% CI 1.02-2.83, p=0.041). No impact of disease risk index, KPS, conditioning regimen, infused CD34 cells on NRM, relapse, RFS or OS was observed. Conclusion: Our study indicates that ALC ≤ 100 is associated with adverse post-transplant outcomes when thymoglobulin dose of 4.5mg/kg is used for in-vivo T cell depletion. This finding may indicate that in pts with lower ALC, thymoglobulin dose may need to be adjusted to optimize its efficacy and avoid toxicities. In the future prospective studies, which evaluate dose reduction of thymoglobulin in pts with low ALC need to be planned to confirm these results. Disclosures Deol: Agios: Other: Advisory board; Novartis: Other: Advisory board; Kite: Other: Advisory board.

scholarly journals Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 581-591 ◽  
Author(s):  
AM Farese ◽  
F Herodin ◽  
JP McKearn ◽  
C Baum ◽  
E Burton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Parameters ◽  
Platelet Recovery ◽  
Hematopoietic Reconstitution ◽  
Radiation Induced ◽  
Complete Blood Counts ◽  
60Co Gamma Radiation

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] 500/microL) and thrombocytopenia (THROM; platelet count 20,000/microL) were assessed. Synthokine significantly (P .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.

scholarly journals Mobilized Peripheral Blood Stem Cells Versus Unstimulated Bone Marrow As a Graft Source for T-Cell–Replete Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide

2017 ◽  
Vol 35 (26) ◽  
pp. 3002-3009 ◽  
Author(s):  
Asad Bashey ◽  
Mei-Jie Zhang ◽  
Shannon R. McCurdy ◽  
Andrew St. Martin ◽  
Trevor Argall ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Peripheral Blood ◽  
Cox Regression ◽  
The United States ◽  
Platelet Recovery ◽  
Post Transplant ◽  
Transplant Outcomes ◽  
Prospective Comparison ◽  
Mobilized Peripheral Blood

Purpose T-cell–replete HLA-haploidentical donor hematopoietic transplantation using post-transplant cyclophosphamide was originally described using bone marrow (BM). With increasing use of mobilized peripheral blood (PB), we compared transplant outcomes after PB and BM transplants. Patients and Methods A total of 681 patients with hematologic malignancy who underwent transplantation in the United States between 2009 and 2014 received BM (n = 481) or PB (n = 190) grafts. Cox regression models were built to examine differences in transplant outcomes by graft type, adjusting for patient, disease, and transplant characteristics. Results Hematopoietic recovery was similar after transplantation of BM and PB (28-day neutrophil recovery, 88% v 93%, P = .07; 100-day platelet recovery, 88% v 85%, P = .33). Risks of grade 2 to 4 acute (hazard ratio [HR], 0.45; P < .001) and chronic (HR, 0.35; P < .001) graft-versus-host disease were lower with transplantation of BM compared with PB. There were no significant differences in overall survival by graft type (HR, 0.99; P = .98), with rates of 54% and 57% at 2 years after transplantation of BM and PB, respectively. There were no differences in nonrelapse mortality risks (HR, 0.92; P = .74) but relapse risks were higher after transplantation of BM (HR, 1.49; P = .009). Additional exploration confirmed that the higher relapse risks after transplantation of BM were limited to patients with leukemia (HR, 1.73; P = .002) and not lymphoma (HR, 0.87; P = .64). Conclusion PB and BM grafts are suitable for haploidentical transplantation with the post-transplant cyclophosphamide approach but with differing patterns of treatment failure. Although, to our knowledge, this is the most comprehensive comparison, these findings must be validated in a randomized prospective comparison with adequate follow-up.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Correction of the Phenotype in Canine Leukocyte Adhesion Deficiency Following Non-Myeloablative, Matched Littermate Transplant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2143-2143
Author(s):  
Yuchen Gu ◽  
Thomas R. Bauer ◽  
Laura M. Tuschong ◽  
Robert A. Sokolic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Disease Phenotype ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Complete Reversal ◽  
Low Levels

Abstract Canine leukocyte adhesion deficiency (CLAD) represents the canine counterpart of the human disease leukocyte adhesion deficiency (LAD). Children with LAD and puppies with CLAD suffer life-threatening bacterial infections as a result of the failure of their leukocytes to adhere to the endothelial surface and migrate to the site of infection. Molecular defects in the leukocyte integrin CD18 molecule are responsible for both LAD and CLAD. Although myeloablative hematopoietic stem cell transplantation can correct the disease phenotype in LAD, this therapy is accompanied by considerable toxicity. Moreover, it is not clear that full donor chimerism is required for reversal of the disease phenotype. To assess the role of mixed chimerism in reversing the disease phenotype in CLAD, we used a non-myeloablative conditioning regimen consisting of 200 cGy total body irradiation preceding matched littermate allogeneic transplant, and followed by a brief post-transplant regimen consisting of cyclosporine and mycophenolic acid. Six dogs received bone marrow cells, three dogs received CD34+ bone marrow stem cells, and four dogs received mobilized peripheral blood stem cells. Eleven of 13 transplanted CLAD dogs achieved mixed donor-host chimerism resulting in complete reversal of the disease phenotype. Donor-derived CD18+ cells measured by flow cytometric analysis in the peripheral blood of the transplanted CLAD dogs correlated closely with donor chimerism measured by DNA analysis of microsatellite repeats in the peripheral blood leukocytes. The 11 dogs with reversal of the CLAD phenotype have been followed for over one year from the time of transplant and displayed levels of donor leukocyte chimerism ranging from 4 to 95%. Since engraftment, all eleven dogs have been free from infection and live in runs with other dogs. Three dogs with very low levels of donor leukocyte chimerism post-transplant displayed evidence of selective egress of CD18+ donor leukocytes into extravascular sites, indicating that the level of CD18+ donor cells measured in the periperal blood may underestimate the total number of CD18+ donor leukocytes. In the two dogs who did not have complete reversal of the CLAD phenotype post-transplant, one dog died at 3 weeks following transplant from a subcapsular hemorrhage of the liver secondary to thrombocytopenia, and one dog had donor microchimerism following transplant with partial reversal of the phenotype. Three dogs who did not have a matched littermate donor, and did not receive a transplant, died of infection at 2, 4, and 6 months of age, respectively. The fact that correction of the CLAD phenotype was achieved in 11 of 13 dogs with mixed donor-host chimerism and the absence of graft-versus-host disease has implications for allotransplant in LAD when a matched sibling donor exists. The observation that very low levels of donor CD18+ leukocytes reversed the disease phenotype supports the use of the CLAD model for testing the ability of autologous, CD18 gene-corrected hematopoietic stem cells to reverse the CLAD phenotype, since low levels of gene correction are anticipated with gene therapy.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

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