Best Approach for Harvesting Bone Marrow to Maximize TNC and CD34+ Cell Counts

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3468-3468
Author(s):  
Nalini K Pati ◽  
Frances Garvin ◽  
Vicki Antonenas ◽  
Ian Kerridge ◽  
Kenneth F Bradstock ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Gvhd ◽  
Cell Number ◽  
Cell Numbers ◽  
Stem Cell Source ◽  
Cell Counts ◽  
Cd34 Cells ◽  
The Right

Abstract Background: Bone marrow (BM) has been utilized as a source of stem cells for transplantation for many years. Although the use of BM has decreased with the advent of mobilized stem cells, utilization is increasing once again due to the lower rate of chronic GVHD associated with BM as a stem cell source. There is no generally accepted technique for harvesting BM. Protocols vary both in relation to the volume of each aspirate, the number of aspirates performed at each puncture site and the total volume of harvests. Method: BM was collected from the posterior iliac crests (PIC) in 2 separate bags: 10ml aspirates from the left and 20 ml aspirates from the right. Samples taken at the start and after 100, 150, 200, 250, & 500 ml were analyzed for TNC, CD34+ and CD3+ cell counts. Results: The following table shows cell number (mean ± SEM ×106) for the parameters indicated. Aspirate Volume (mls) Parameter Start (n=4) 100mls (2) 150mls (2) 200mls (2) 250mls (4) 500mls (4) 10 TNC 555 ± 28 286 ± 38 257 ± 40 226 ± 8 199 ± 11 158.5±18.5 CD34 5.8 ± 0.05 1.8 ± 0.1 1.7 ± 0.2 1.3 ± 0.4 1.1 ± 0.2 0.8 ± 0.1 CD3 65.8 ± 12.0 35.8 ± 6.9 32.8 ± 8.0 29.9 ± 1.5 28.4 ± 5.1 29.8 ± 6.2 20 TNC 914 ± 52 627 ± 137 458 ± 44 429 ± 113 391 ± 81 264 ± 24 CD34 9.1 ± 0.5 3.8 ± 0.2 2.4 ±0.2 2.7 ± 0.1 2.3 ± 0.7 1.0 ± 0.2 CD3 106.9 ±22.1 64.6 ± 5.3 55.0±14.1 56.5±14.7 53.7±14.6 41.2 ± 9.6 There is a rapid fall in the yield of CD34+ cells obtained with increasing harvest volume (19 and 25% of the initial number after 250 ml for 10 and 20 ml aspirates respectively; 14 and 11% respectively after 500 ml). In contrast the CD3+ cell numbers fall more slowly (43 and 50% after 250 ml, 45 and 38% after 500 ml). By the time 500 ml has been aspirated, there is no difference in the total number of CD34+ cells obtained from a 10 ml versus a 20 ml aspirate of bone marrow. Conclusion: CD34+ cell yields fall rapidly when BM is harvested along the PIC. Using additional areas such as the anterior iliac crests may be preferable to a large volume PIC harvest for optimizing CD34+ stem cell collection. After 500 ml of BM has been harvested, 20 ml BM aspirates do not increase CD34+ cell numbers and 10 ml aspirates should be taken to minimize unnecessary blood loss and reduce T cell contamination.

Radiation Dose Determines Degree of Donor Chimerism after Nonmyeloablative Hematopoietic Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1828-1828 ◽  
Author(s):  
Christoph Kahl ◽  
Marco Mielcarek ◽  
Mineo Iwata ◽  
Michael Harkey ◽  
Barry Storer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Radiation Dose ◽  
Low Dose ◽  
Donor Chimerism ◽  
Stem Cell Source ◽  
Cd34 Cells ◽  
Cell Source ◽  
Total Body

Abstract Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivo irradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.

Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2846-2846
Author(s):  
M. Kozik ◽  
J. Banks ◽  
L. Fanning ◽  
M. Finney ◽  
Y. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Flow Analysis ◽  
Scid Mice ◽  
Mouse Bone Marrow ◽  
Short Term ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3106-3106
Author(s):  
Pietro Sodani ◽  
Buket Erer ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
Andrea Roveda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Marrow Transplant ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Increased Aldehyde Dehydrogenase Activity Indentifies a Subset of Human Leukemic Blasts with Stem Cell Characteristics and Correlates with Poor Prognosis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1345-1345
Author(s):  
Dan Ran ◽  
Mario Schubert ◽  
Larissa Pietsch ◽  
Isabel Taubert ◽  
Christian Wallenwein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Prognostic Factors ◽  
Aldehyde Dehydrogenase ◽  
Protein Level ◽  
Aldh Activity ◽  
Cd34 Cells ◽  
Dividing Cells

Abstract INTRODUCTION: Normal hematopoietic stem cells (HSC) are characterized by their ability to self-renew, to generate multiple cell-lineages, and show slow divisional kinetics. Leukemic stem cells (LSC) have been reported to show similar characteristics but their identification has been elusive. We have studied various methods and have identified aldehyde dehydrogenase (ALDH) staining as an optimal method for the enrichment of primary human LSC. MATERIAL&METHODS: Bone marrow samples were obtained from patients with newly diagnosed AML after informed consent. Mononuclear cells were stained with Aldefluor and sorted by flow cytometry according to their forward/side scatter characteristics and ALDH activity (ALDH+/ALDH−). Alternatively, primary AML samples were being enriched for CD34+ cells by magnetic column, then double-stained with CD34-antibodies and Aldefluor and sorted for the co-expression of CD34+ and ALDH+, respectively for CD34+ alone. Human Mesenchymal Stromal Cells (MSC), isolated from human bone marrow, were used as a surrogate model for the cellular microenvironment of the hematopoietic niche. Adhesion of various AML cell lines and subpopulations of primary leukemic cells (ALDH+, ALDH−, CD34+, CD34+/ALDH+, all blasts) to MSC was tested in the adhesion chamber assay. Semi-quantitative RT-PCR was used to analyze the gene expression of various adhesion molecules and Western- Blot analysis was performed to validate the PCR-results on protein level. The generation of secondary leukemic colonies was evaluated in a semi-solid methylcellulose medium, as well as in a long term co-culture system (LSC-IC assay; in analogy to the LTC-IC assay). RESULTS: The percentage of ALDH+ cells ranged from 0.01% to 13.2% with a median of 1.47% (n=55). Adhesion significantly differed in the ALDH+ and ALDH− subpopulations: 85±4% of ALDH+ cells but only 61±8% of ALDH− cells were adherent (n=11, p<0.001). Adhesion molecules, such as CXCR4 and CD44, were highly expressed on the ALDH+ subpopulation both on mRNA level and protein level, in contrast to the ALDH− subpopulation. Analysis of the initial divisional kinetics on single cell base showed that the ALDH+ subpopulation contained more slow dividing cells whereas the majority of the ALDH− subpopulation consisted of fast-dividing cells (n=3; p<0.01). The frequency of long term leukemic colony initiating cells (LSC-IC) was 3.82% in the ALDH+ but only 0.01% in the ALDH− (n=21; p<0.01). In the CD34+ the LSC-IC frequency was 1.96% versus 0.01% in the CD34− (n=5, p<0.01). The highest LSC-IC frequency could be monitored in ALDH+/CD34+ cells: 6.1% generated secondary leukemic colonies (n=5). These colonies, harvested after 7 weeks of cultivation, were examined for their immune phenotype and screened for cytogenetic aberrations by fluorescent in situ hybridization (FISH) and the chromosomal aberrations were consistent with the AML samples taken at diagnosis. Furthermore, the frequency of ALDH+ cells correlated significantly with adverse prognostic factors: patients with a high-risk karyotype had a mean of 2.9% ALDH+ cells (n=21); in contrast, patients with a normal karyotype had a mean of 0.4% ALDH+ cells in their bone marrow (n=34; p<0.001). The ability of ALDH+ versus ALDH− subsets to generate secondary leukemia in the animal model is concurrently examined. DISCUSSION: In summary, measurement of the ALDH activity provides a useful tool for the isolation of a distinct AML-blast subpopulation with stem-cell like features (LSC). The ALDH+ subsets showed higher affinity to the surrogate niche (MSC), elevated expression of CD44, Cadherin-2, and CXCR4 and were associated with an increased frequency of secondary leukemic colonies in vitro (LSC-IC). Above all, the frequency of ALDH+ blasts correlated with clinical prognostic factors, which substanciates LSC as a relevant therapeutic target.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Peripheral Blood Stem Cells Vs Bone Marrow: Stem Cell Source Comparison for Patients Acute Myeloid Leukemia and Myelodysplastic Syndrome Receiving an Allogeneic Stem Cell Transplantation from a 10/10 Matched Donor. Study from the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2552-2552 ◽  
Author(s):  
Aurélie Ravinet ◽  
Aurélie Cabrespine ◽  
Gerard Socie ◽  
Noel Milpied ◽  
Ibrahim Yakoub-Agha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Disease Free Survival ◽  
French Society ◽  
Peripheral Blood Stem Cells ◽  
Cell Therapies ◽  
Gvhd Prophylaxis

Abstract Introduction: Peripheral blood stem cells (PBSC) are increasingly used for unrelated donor (UD) hematopoietic stem cell transplantation (HSCT). A recent randomized prospective trial did not detect significant survival differences between PBSC and bone marrow (BM) transplantation from a UD. The use of PBSC reduced the risk of graft failure, whereas BM reduced the risk of chronic graft versus host disease (GVHD) (Anasetti & al, NEJM 2012). However, HLA matching was based on HLA-A, HLA-B, HLA-C &HLA-DRB1 (8/8 but also 7/8), some of the patients (pts) received a reduced intensity conditioning regimen (22%) and diseases consisted of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), but also other hematological malignancies. We thus conducted this study to compare mobilized PBSC with BM for matched 10/10 UD HSCT after standard conditioning regimen (MAC) in pts with AML and MDS only. Patients and methods: We included all consecutive pts in France who received a first HSCT for AML or MDS with PBSC or BM from a matched 10/10 UD after a MAC (fractionated total body irradiation (TBI)-Cyclophosphamide(Cy), n=165 or Endoxan-Cy, n=203) between 2000 and 2013. Clinical data were prospectively collected using ProMISe (Project Manager Internet Server), an internet-based data registry system shared by all centers of the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC). HLA typing data were collected from the SFHI (French Society of Histocompatibility and Immunogenetic) and the ABM (French Biomedical Agency). This study is in accordance with Helsinki declaration for clinical research. Results: We included368 adults pts (221 [60%] received BM; 147 [40%] received PBSC). Median follow-up was of 16.5 months [0-156]. The BM and PBSC groups were well balanced with respect to age, diagnosis, disease risk, use of antithymocyte globulins (ATG) (67 [30%] BM and 52 [35%] PBSC recipients,) and cytomegalovirus (CMV) recipient and donor status. PBSC recipients were more likely to be male and received less TBI-based regimen. GVHD prophylaxis mainly combined cyclosporine A (CSA) and methotrexate (MTX) (82%). The median number of nucleated cell dose infused was higher in the PBSC group compared with the BM group: CD34+ cells, 6.96 x10⁶/kg [1.2-37.8] vs 2,78 x10⁶/kg [0.6-89] p<0.01) and total nucleated cells, 9.8 x10⁸/kg [1.3-663] vs 2.3 x10⁸/kg [0.3-305.3] p<0.01). Two hundred and seven (90%) pts engrafted after BM and 144 (99.3%) after PBSC HSCT (p=0.1). Among pts who received PBSC as compared with those who received BM, the median time to neutrophils engraftment (> 0.5 x 109 /L) was 6 days shorter and 8 days shorter to platelets engraftment (>20x109 /L) (p<0.01). The cumulative incidence (CI) for severe acute GVHD III-IV was 21.1% and 16.3% in the PBSC and the BM group, respectively (p=0.18). CI of chronic GVHD was higher after PBSC (47.1% vs 34.3% for BM, p=0.05). By multivariate analysis, the absence of ATG in the conditioning regimen (HR 0.4 95%CI [0.22-0.72] p<0.01) and PBSC as stem cells source (HR 0.6 95%CI [0.34-0.97] p=0.04) were associated with an increased chronic GVHD. At 2-years, the CI of non relapse related mortality (NRM), relapse as well as disease free survival (DFS) and overall survival (OS) were similar between the 2 groups (Table 1). In multivariate analysis, better OS was associated with complete remission (CR) disease status (HR 0.5 95%CI [0.32-0.69] p<0.01) and pts’s age<38.1 years (HR 0.67 95%CI [0.48-0.93] p=0.02) at time of HSCT, and the use of CSA-MTX as GVHD prophylaxis (HR 0.6 95%CI [0.41-0.95] p=0.03). Conclusion: OS, NRM and relapse rates are similar with PBSC and BM after HLA 10/10 matched UD for AML or MDS using MAC, but engraftment is better with PBSC and the CI of chronic GVHD is lower with BM. Better results are obtained for pts <38 years old with a disease in CR at time of HSCT using CSA-MTX as GVHD prophylaxis. The absence of ATG with PBSC was associated with chronic GVHD. This study thus favors the use of ATG in the setting of matched 10/10 PBSC. However, the role of ATG in the context of BM after HLA 10/10 matched UD MAC HSCT remains unclear and warrants further investigation. Table 1: 2-year CI of NRM*, relapse, DFS** and OS*** Parameters BM % (95% CI) PBSC % (95%CI) p value NRM 23 (20-26) 18 (15-21) 0.8 Relapse 30 (27-33) 28 (25-31) 0.83 DFS 47.1 (44-51) 54.4 (50-58) 0.2 OS 54 .4 (50.8-57.9) 60.2 (55.7-64.6) 0.31 *NRM: non-relapse mortality **DFS: disease free survival ***OS: overall survival Disclosures No relevant conflicts of interest to declare.

The Best of Both? Megadose Stem Cells with CD34 Selection and T-Cell Addback Enables Engraftment from Mismatched Unrelated Donors Using Reduced Toxicity Conditioning in Primary Immunodeficiency

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4303-4303
Author(s):  
Juliana Montibeller Silva ◽  
Kanchan Rao ◽  
Robert Chiesa ◽  
Stuart Adams ◽  
Margaret Brocklesby ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
T Cell ◽  
Primary Immunodeficiency ◽  
Chronic Gvhd ◽  
Unrelated Donor ◽  
Peripheral Blood Stem Cells ◽  
Stem Cell Source ◽  
Reduced Toxicity

Abstract Background: The optimal approach for transplanting patients with primary immunodeficiency from a mismatched unrelated donor (MMUD) remains unclear. With reduced intensity conditioning, when bone marrow (BM) was used as the stem cell source in such patients we have previously observed a high rate of very low level donor chimerism (4 of 12 patients) requiring a second transplant procedure. The use of peripheral blood stem cells (PBSC) can overcome this but results in a high incidence of acute (≥ Gde II 10/21, Gde III-IV 5/21) and chronic (10/21, extensive 7/21) graft-versus-host disease (GVHD) (Rao et al in press). We hypothesized that the use of megadose CD34 selected stem cells from PB with addback of a T-cell dose akin to BM might facilitate engraftment through improved competition for the stem cell niche without severe GVHD. Methods: We prospectively analysed outcomes on 20 consecutive patients with primary immunodeficiency (SCID n=5, HLH n=3, Other PID n=12) transplanted from 8/10 (n=4) or 9/10 (n=16) HLA MMUD at our institution between 2011-2015. The mean age at transplant was 5.1 years. All patients received reduced toxicity conditioning regimens (12/20 Fludarabine/Melphalan/Alemtuzumab; 8/20 Fludarabine/Treosulphan/Alemtuzumab) with Cyclosporin (CsA) and mycophenolate mofetil (MMF) GvHD prophylaxis. CD34 selection of donor PBSC was performed using CliniMACs and the mean CD34+ dose was 20.1x106/kg. At day 0 either 108 CD3/kg (cohort 1, n=6) or 3x108 CD3/kg (cohort 2, n=14) T-cells from the CD34- fraction were infused with the graft. Mean follow up was 31.9 months. Results: In cohort 1, all patients engrafted and 2 developed high level donor mixed chimerism (both curative) in the myeloid and lymphoid lineages at last follow up. Two patients had Grade II acute GvHD and 1 had moderate chronic GvHD. In view of the slow immune reconstitution in this cohort, the T-cell addback dose was increased to 3x108/Kg in subsequent patients. In cohort 2, all patients achieved full donor haemopoiesis initially, 6/14 developed high levels of donor chimerism in both myeloid and lymphoid lineages later and 1/14 progressed with 10% donor T-cell engraftment but remains disease-free. The incidence of significant aGVHD was low (grade II n=4, no grade III or IV) and no patient developed cGvHD. Overall across both cohorts, 10 patients had viral reactivations and there were 5 deaths (3 viral complications, 1 pulmonary vasculopathy, 1 cGVHD lungs). The disease free survival at 2 years 7 months follow up was 70% which compares favorably with a previous cohort transplanted with unmanipulated BM as the stem cell source (Fig 1). Immune reconstitution was delayed (mean CD3 and CD19 counts at day 100 post-transplant was 245 x 109/L and 315 x 109/L) with similar kinetics in both cohorts (Fig 2), comparable to RIC transplant using unmanipulated BM. By 1 year post transplant 11/14 evaluable patients achieved normal CD3+T-cell numbers and 8/14 normal CD19+ B-cell counts. Conclusion: The use of megadose peripheral blood stem cells with T-cell addback in the mismatched unrelated donor transplant setting results in high rates of curative engraftment and a low incidence of acute and chronic GvHD following reduced toxicity conditioning. However, T-cell reconstitution remains delayed (presumably reflecting the effect of Alemtuzumab on the infused T-cells) with a high incidence of viral complications. Disclosures No relevant conflicts of interest to declare.

scholarly journals UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2500-2500
Author(s):  
Tellechea Maria Florencia ◽  
Flavia S. Donaires ◽  
Tiago C. Silva ◽  
Lilian F. Moreira ◽  
Yordanka Armenteros ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Bone marrow mesenchymal stem cell transplantation in acute brain trauma

2013 ◽  
Vol 52 (05) ◽  
pp. 192-197 ◽  
Author(s):  
B.-N. Park ◽  
J.-K. Yoon ◽  
Y.-S. An
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Treatment Group ◽  
Glucose Metabolism ◽  
Brain Trauma ◽  
Fdg Uptake ◽  
18F Fdg ◽  
Stem Cell Treatment ◽  
The Right

SummaryAim: This study was performed to evaluate the effects of intravenously transplanted rat bone-marrow derived mesenchymal stem cells (rBMSCs) in an acute brain trauma model using serial 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in rat models. Animals, methods: Trauma models were made using a controlled cortical impact injury device. The stem cell treatment group was treated with intravenous injections of BMSCs, and models without stem cell therapy comprised the control group. Serial 18F-FDG PET images were obtained 1, 7, 14, 21, and 28 days after trauma. The difference in 18F-FDG uptake between day 1 and each time point after trauma was analyzed with SPM2 (uncorrected p < 0.005). Results: The stem cell treatment group demonstrated significantly higher 18F-FDG uptake in the right parietal region at 14 days after trauma than at 1 day after trauma. An increase in glucose metabolism in the right parietal cortex appeared on days 21 and 28 after trauma in the group without stem cell treatment. The 18F-FDG uptake in the brain was improved over a broader area, including the right parietal and right primary somatosensory cortex, on days 21 and 28 after trauma in the stem cell treatment group compared with the group without stem cell treatment. Conclusion: BMSC therapy in trauma models led to improved glucose metabolism. This result might support the therapeutic effect of stem cells in brain trauma.

Clinical Outcomes of Cord Blood Transplantation from Unrelated Donors Comparable with Marrow or Blood Transplantation from Related Donors in Adults: A Single Institute Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 306-306
Author(s):  
Satoshi Takahashi ◽  
Jun Ooi ◽  
Akira Tomonari ◽  
Takaaki Konuma ◽  
Kenji Fukuno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Acute Gvhd ◽  
Cord Blood Transplantation ◽  
Chronic Gvhd ◽  
Blood Transplantation ◽  
Stem Cell Source ◽  
Cell Source ◽  
Unrelated Donors

Abstract We previously reported some promising results of cord blood transplantation (CBT) compared with bone marrow transplantation (BMT) from unrelated donors in terms of graft-versus-host disease (GVHD), transplant-related mortality (TRM), and disease-free survival (DFS) in our institute (Blood104: 3813, 2004). If the patient was eligible for allogeneic transplantation without any related donors, we performed CBT immediately, rather than waiting for the results of an unrelated marrow donor search. This might be one of the reasons for our favorable CBT results in adults compared with most previously published studies. We studied the clinical outcomes of 163 adults with hematological malignancies who received unrelated CBT (n=92), or BMT or peripheral blood stem cell transplantation (PBSCT) from related donors (n=71, 55 BMT and 16 PBSCT) between January 1997 and February 2005. All patients received myeloablative regimens including 12 Gy of total body irradiation and almost the same supportive care. We analyzed the hematopoietic recovery, rates of GVHD, risks of TRM and relapse, and DFS using Cox proportional hazards models. The age, sex, cytomegalovirus serological status, time from diagnosis to transplantation, and GVHD prophylaxis regimens were not significantly different between both groups. Overall rates of high-risk patients in the CBT and in BMT/PBSCT groups were 58% and 63%, respectively. Human leukocyte antigen (HLA) was scored serologically for HLA-A and B and genetically for DRB1 alleles. There were no complete matches in CBT and 54 (76%) matched grafts in BMT/PBSCT. Median numbers of leukocytes and CD34+ progenitor cells before freezing of cord blood grafts were 2.4x107/kg and 0.9x105/kg, respectively. Median follow-up was 27 months for CBT and 50 months for BMT/PBSCT. Significant delays in neutrophil and platelet engraftment rates occurred after CBT; however, overall myeloid engraftment rates were almost the same for both grafts (94% in CBT and 98% in BMT/PBSCT). The cumulative incidences of grades II to IV acute GVHD, of grades III and IV acute GVHD, and of requiring steroids for treating acute GVHD among CBT recipients were 58%, 8%, and 18%, respectively. Those among BMT/PBSCT recipients were 58%, 19%, and 38%, respectively. Chronic GVHD affected 68 of 75 CBT and 49 of 60 BMT/PBSCT evaluable recipients. Twenty-two CBT and 30 BMT recipients developed extensive GVHD. The 1-year cumulative incidence of TRM, the 3-year cumulative incidence of relapse, and the 3-year probability of DFS in CBT recipients were 9%, 18%, and 71%, compared with 13%, 26%, and 60% in BMT/PBSCT recipients. Multivariate analysis demonstrated no apparent difference in those outcomes between both groups. Taken together, engraftment speed was slower and severe acute GVHD and extensive chronic GVHD tended to be lower in CBT recipients compared with BMT/PBSCT recipients; however TRM, relapse and DFS were comparable in both groups. These data suggest that cord blood from unrelated donors could be as safe and effective a stem cell source as bone marrow or mobilized peripheral blood from related donors for adults when it is used as a primary unrelated stem cell source.

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