Purified T Depleted Peripheral Blood and Bone Marrow CD34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5161-5161
Author(s):  
Pietro Sodani ◽  
Marco Andreani ◽  
Paola Polchi ◽  
Javid Gaziev ◽  
Filippo Centis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Preparative Regimen ◽  
Chain Synthesis

Abstract Approximately 60% of thalassemic patients can not apply to “gene terapy 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 estabilished 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 nine thalassemic children aged 3 to 8 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 untill 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 sistem), 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) prophilaxis. Four patients rejected the transplant and are alive with thalassemia: one patient received a different dose of CD3 without cyclosporine after transplant, two patients received a lower dose of CD34+, in the fourth patient the donor has been the haploidentical father instead than the mother. One of the nine patients, after the failure of the transplant from the mother, received a second transplant using purified CD34+ cells from the father, using the same preparative regimen and achieved a complete hematopoietic reconstitution. Six patients are alive disease free with a median follow up of 19 months (range 7–30). None of the six patients showed AGVHR. 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 whithout genotipically or phenotipically HLA identical donor.

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.

scholarly journals Choice of Unmanipulated T Cell Replete Graft for Haploidentical Stem Cell Transplant and Posttransplant Cyclophosphamide in Hematologic Malignancies in Adults: Peripheral Blood or Bone Marrow—Review of Published Literature

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Shatha Farhan ◽  
Edward Peres ◽  
Nalini Janakiraman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Hematologic Malignancies ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Cell Sources

Allogeneic hematopoietic stem cell transplantation (SCT) is often the only curative option for many patients with malignant and benign hematological stem cell disorders. However, some issues are still of concern regarding finding a donor like shrinking family sizes in many societies, underrepresentation of the ethnic minorities in the registries, genetic variability for some races, and significant delays in obtaining stem cells after starting the search. So there is a considerable need to develop alternate donor stem cell sources. The rapid and near universal availability of the haploidentical donor is an advantage of the haploidentical SCT and an opportunity that is being explored currently in many centers especially using T cell replete graft and posttransplant cyclophosphamide. This is probably because it does not require expertise in graft manipulation and because of the lower costs. However, there are still lots of unanswered questions, like the effect of use of bone marrow versus peripheral blood as the source of stem cells on graft-versus-host disease, graft versus tumor, overall survival, immune reconstitution, and quality of life. Here we review the available publications on bone marrow and peripheral blood experience in the haploidentical SCT setting.

scholarly journals Low Dose Umbilical Cord Blood Transplant Results in Skewed Immune Cell Composition Which May Impact Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5672-5672
Author(s):  
Chi Hua Sarah Lin ◽  
Beth Shaz ◽  
Rona Singer Weinberg
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Low Dose ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Introduction Reconstitution of donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) is essential for recovery and long-term survival. Despite routine use of human umbilical cord blood (hUCB) as a stem cell source for allogeneic HSCT, much remains unknown regarding the kinetics of immune recovery and correlation with different transplant cell dosages. To study the hUCB repopulating potential, different hUCB CD34+ cell dosages were transplanted into immune deficient NSG mice; hematopoietic cells were then collected and engraftment was analyzed. Methods NOD/SCID/IL-2Rγnull recipient (NSG) mice (Jackson Laboratories, Bar Harbor, ME) were kept in pathogen-free facilities. CD34+ cells were isolated from a pool of six hUCB donors using a CD34+ microbead kit (Miltenyi Biotec). Each sublethal irradiated (220 or 300 cGy) 8 week old female NSG mice received either low dose (15x103, N=15) or high dose (75x103, N=15) CD34+ cells transplanted intravenously via retro-orbital route. Animal experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines. Statistical analysis was performed with Prism software (GraphPad Software, Inc) and Excel. Data are presented as mean ± standard error of the mean (SEM). Results To determine the effects of hUCB CD34+ cell dosages on the rate of engraftment, NSG mice were transplanted with low doseor high dose CD34+ cells. The transplanted CD34+ cell dosages were comparable to clinical dosages based on body weight (Mavroudis et al. 1996). The engrafted cells were analyzed for expression of surface markers that define human hematopoietic cells. During the follow up period of up to 18 weeks, the high dose infused group had increased hUCB engraftment compared with the low dose infused group in peripheral blood (Fig 1A), bone marrow (Fig 1B & 1C) and spleen (Fig 1D), which is consistent with reported clinical observations that infused cell dosage is inversely correlated with time to engraftment (Migliaccio et al. 2000 Blood). Interestingly, we observed different lymphoid subset frequencies between low and high dose infused groups at the post-engraftment stage (18 weeks post transplantation) (data not shown). To investigate different lymphoid subset engraftment frequencies in low and high dose hUCB transplanted recipient mice at early engraftment stage, peripheral blood and hematopoietic organs were collected and analyzed up to 10 weeks post transplantation. The low dose infused group had significantly lower CD3+ (T cells) and CD56+ (NK cells) frequency in peripheral blood at 4 and 8 weeks (Fig 2A & 3A). More importantly, CD3+ (T cells) frequency was close to non-detectable in the bone marrow and spleen in the low dose infused group (Fig 2B & 2C), and CD56 (NK cells) frequency was decreased in the low dose infused group compared with the high dose infused group (Fig 3B & 3C). The absolute CD3+ and CD56+ number, displayed as fold difference, were even more dramatically decreased in the femur (Fig 2D & 3D) and the spleen (Fig 2E & 3E) of low dose infused group. Because of the substantial difference in T cell subset frequencies between the two dosage groups in bone marrow and spleen, thymuses were collected and analyzed to study T cell development and maturation. Engraftment of hCD45+ cells in the thymuses were observed in 10 out of 15 animals (66.7%) in the low dose infused group and 12 out of 14 animals (85.7%) in the high dose infused group. Interestingly, in animals with high hCD45+ frequency, the total thymocyte CD3+ frequency was lower in the low dose infused group (Fig 4A). Additionally, the low dose infused group had lower CD3+CD4+ T cell frequency (Fig 4B) and higher CD3+CD4+CD8+ T cell frequency (Fig 4C), suggesting low dose infused group had a decreased mature T cell population and increased immature T cell population in the thymus. In contrast, the low dose hUCB CD34+ cells infused group had increased hCD19 (B cells) frequency in the peripheral blood, bone marrow and spleen (Fig 5A-5C), while the absolute hCD19 (B cells), displayed as fold difference, did not show a statistically significant difference between the two groups (Fig 5D & 5E). Conclusions In summary, our findings suggest that (1) transplanted hUCB cell dosage is inversely correlated with time to engraftment (2) low transplanted hUCB cell dosage resulted in skewed immune cell population which may contribute to delayed immune recovery after allogeneic HSCT. Disclosures No relevant conflicts of interest to declare.

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.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Generation of Human T/NK Cell Precursors for Adoptive Transfer To Enhance Immune Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3209-3209
Author(s):  
Sonali Chaudhury ◽  
Johannes Zakarzewski ◽  
Jae-Hung Shieh ◽  
Marcel van der Brink ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with significant post-transplant immunoincompetence which affects in particular the T cell lineage and results in an increased susceptibility to infections. Novel strategies to enhance immune recovery after HSCT could prevent malignant relapse and immune deficiency and improve the overall outcome of this therapy. We have established a serum free culture system using murine bone marrow stroma expressing the Notch ligand Delta-like 1 (DL1) to obtain high numbers of human pre-T cells from CD34+ cells. Human cord blood CD34+ cells were plated on OP9 DL1 stroma transduced with adenovirus expressing thrombopoietin (ad-TPO) at an MOI of 30. Media used was QBSF-60 (Serum free media prepared by Quantity Biologicals) supplemented with Flt-3 ligand and IL-7 (10ng/ml). At 4–5 weeks we obtained a 10 5–10 7 fold expansions of cultured cells of which about 70–80% were CD5, CD7 positive pre T cells (Fig 1). We then developed an optimal system to study human lymphohematopoiesis using mouse models (NOD/SCID/IL2rϒnull and NOD/SCIDβ2null) and established an adequate pre T cell number (4 × 10 6) and radiation dose (300 Rads). We injected CD34 and pre-T cells (CD45 +, CD4−, CD5+, CD7+) derived from OP9 DL1 cultures into these mice and achieved ~50%engraftment of NK in the bone marrow and spleen of the mice at 2 weeks following transplant. The thymus from the same mice showed evidence of about 12–15% CD7+ pre T cells. We are currently studying the function of the generated NK and T cells both in vivo and in vitro studies. Figure Figure

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Haplo-Identical Allogeneic Stem Cell Transplantation Using GCSF-Mobilized Marrow Plus Blood Stem Cells from Parent Donors for Children with High-Risk Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5084-5084
Author(s):  
Quanyi Lu ◽  
Xiaoqing Niu ◽  
Peng Zhang ◽  
Delong Liu
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Risk ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells

Abstract Increasing number of patients in China have difficulty of finding sibling donors due to limited number of siblings. We therefore explored the feasibility using haploidentical parent donors for allogeneic hematopoietic stem cell transplantation. Eight leukemia patients were studied in our hospital. These included 2 CML-BC, 2 MDS-RAEB, 3 relapsed ALL and 1 relapsed AML. The median age was 12 (7–17). GCSF- mobilized bone marrow and peripheral blood stem cells were collected from parents (1 to 3 locus mismatched). The conditioning regimen consisted of fludarabine (30mg/m2/d x5), bulsulfan (4mg/kg/d x3) and cyclophosphamide (50mg/kg/d x2). Cyclosporin A, mycophenolate mofetil, methotrexate, and ATG were used for GVHD prophylaxis. The total number of CD34+ cell in the grafts ranged between 5–10 x 106/kg. The median follow- up was 13 months (6–24). One patient failed to engraft, the other 7 patients achieved full donor chimerism at day 28. The incidence of acute GVHD (grade II-IV) was 57.1% (4 of 7). The incidence of chronic GVHD of limited stage occurred in the same 4 patients. One patient died of lung complication at 17th month, another patient with CML-BC relapsed 10 months after transplantation. The rest 6 patients are alive without disease. These results suggested that parents could be considered as stem cell donors in the absence of alternative donors for young patients with high-risk diseases. GCSF-primed bone marrow plus peripheral blood stem cells might be beneficial to reduce the risk of GVHD for leukemia children in China. More patients are needed to further study this approach.

The “MarrowMiner”: A Novel, Minimally Invasive Device for the Harvest of Bone Marrow: Initial Human Experience Reveals Safety, Efficacy and Richer Cell Product Than Standard Harvest Methods

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4130-4130
Author(s):  
Daniel L. Kraft ◽  
Vartan Ghazarossian ◽  
Mike Crocker ◽  
Sergio Najar ◽  
Antonio A. Carrasco-Yalðn
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell ◽  
Minimally Invasive ◽  
Progenitor Cells ◽  
Local Anesthesia ◽  
Peripheral Blood ◽  
Single Entry

Abstract INTRODUCTION: Bone marrow (BM) contains a rich supply of adult stem and progenitor cells, including hematopoieitic and mesenchymal stem cells which are used in Bone Marrow Transplantation (BMT) and an increasing array of regenerative therapies. Traditional marrow harvest methods utilize percutaneous large bore needle aspiration, result in marrow highly diluted by peripheral blood, and are crude, tedious, labor intensive and expensive, usually requiring general anesthesia, and >100 serial small volume aspirates to obtain adequate cell numbers for BMT. BM is showing increasing long-term advantages over mobilized PBSC for many alloegeneic BMTs, in terms of less cGVHD and in some cases improved survival. Improved BM harvest methods are needed. A novel device, the “MarrowMiner” (MM), was developed for the minimally invasive harvest of BM to enable the rapid, convenient, outpatient harvest of large quantities of BM under local anesthesia for use in allogeneic and autologous BMT and cell therapies utilizing autologous marrow derived cells. The MarrowMiner utilizes a single marrow entry site into the anterior or posterior iliac, through which the flexible, powered, guidable FlexShaft catheter can access the majority of the marrow space and aspirate rich marrow. Extensive testing in human cadavers and porcine models demonstrated a 10X increase in stem cells activity/ml (by CFU) compared to that of traditional needle harvests. The MM recently received both FDA and CE Mark regulatory approved, and ‘First In Human’ trials were successfully completed under local anesthesia, demonstrating safety, efficacy and higher stem cell yields compared to traditional methods. METHODS: In an ongoing prospective study, 10 patients undergoing autologous marrow derived therapy for use in regenerative medicine, had marrow harvested from their anterior or posterior ileac by the MM under local anesthesia on one hip, with direct comparison to standard needle serial marrow aspirates on the patients opposite hip (up to 350 ml per side). Cell viability, counts, CD34+, T cell, and MSC populations were assessed by flow cytometry. RESULTS: The MM successfully harvested marrow from a single entry sites and 2–3 paths under local anesthesia, without complications. Compared to standard harvest in the same patients, MM harvests had significantly number of Total Nucleated Cells ml compared to marrow harvested from the same patient by standard needle ( mean 1.98 fold greater TNC (range 0.87–3.36, p<.05). Viability was equivalent at (>99). In addition to higher TNC/ml, significantly higher levels (mean 3.56 fold) of Aldeflour/ALDH+ cells/ml, CD34+, and phenotypic MSC (CD45−,34−,90+,105+) and endothelial progenitor cells were obtained, as measured by flow cytometry. Mean CD3+ T-cell counts per ml were lower with MM harvests. CONCLUSIONS: The novel FDA approved MarrowMiner system demonstrated safety and efficacy in clinical use, harvesting more stem cells per unit volume in a single entry compared to standard harvest methods. These results suggests the MM may enable improved clinical stem cell harvests in a more rapid and minimally invasive manner in the outpatient setting, while harvesting a richer marrow product with less peripheral blood contamination. Such a system, facilitating convenient, on demand stem cell collection may have significant application for BMT and other marrow based cellular therapies.

Poor Graft Function in Recipients of T Cell Depleted (TCD) Allogeneic Hematopoietic Stem Cell Transplants (HSCT) Is Mostly Related to Viral Infections and Anti-Viral Therapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3147-3147 ◽  
Author(s):  
Roni Tamari ◽  
Sheetal Ramnath ◽  
Deborah Kuk ◽  
Craig S. Sauter ◽  
Doris M Ponce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Viral Infections ◽  
Graft Function ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Poor Graft Function

Abstract Abstract 3147 Introduction: Poor graft function (PGF) without immune rejection, defined as persistent cytopenias with hypocellular marrow and full donor myeloid chimerism, can be a life-threatening complication after allogeneic HSCT. It is commonly caused by viral infectious, myelosuppressive drugs like antivirals, and graft-vs-host disease (GvHD). Treatment options include supportive therapy with transfusions and growth factors and in severe cases administration of additional hematopoietic stem cells (HSCs) from the same donor without conditioning (stem cell boost). The incidence, natural history, and the indications for stem cell boost therapy are not well defined. Aims: To assess the incidence, etiologies, and indications for stem cell boost for PGF in a homogeneous group of patients with advanced MDS and AML who underwent TCD HSCT from matched or mismatched related or unrelated donors after conditioning with the same myeloablative regimen. Patients and methods: Poor graft function was defined as persistent neutropenia (ANC <1,000 μL and G-CSF administration x3 in 30 days), thrombocytopenia (platelets <50,000 μL or platelets transfusion × 4 in 30 days), and/or hemoglobin <8 g//dL after engraftment with hypocellular BM and full donor myeloid chimerism. Severe PGF was defined as ANC <500 μL, red cell transfusion-dependent anemia with reticulocytopenia of < 20,000 μL, and platelets <20,000 μL. The patient population in which this study was done included 42 patients enrolled between 09/2009 and 05/2012 in a phase 2 trial of palifermin peri-transplant to reduce transplant-related mortality. The median age was 57.5 years (1–65). All patients received the same myeloablative conditioning regimen with busulfan, melphalan, fludarabine, rabbit ATG and palifermin peri-transplant. G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Donors were HLA matched (31; 13 related and 18 unrelated) or mismatched unrelated (11). Chimerism was determined in bone marrow as well as neutrophils, B cells, and T cells by short tandem repeat analysis on DNA extracted from bone marrow and peripheral blood cell subsets. Results: Forty-one patients were evaluable for this analysis; 1 patient was not included as he rejected the allograft shortly after engraftment. There were 8 cases of PGF with a cumulative incidence (CI) at 1 year of 18% (13% HLA matched, 33% HLA mismatch). The etiology was infection in 7 cases, and unknown in the 8th case. This patient presented with presumed autoimmune anemia and thrombocytopenia associated with a hypercellular marrow and did not respond to multiple lines of therapies. Her marrow became later hypocellular and met the criteria for PGF. None of the PGF cases in this series was associated with GvHD at the time of diagnosis of PGF. The infectious etiologies included: 6 viral infections and 1bacterial sepsis + myelosuppressive drugs. The most common viral etiology associated with PGF was CMV (50%). The 1-year CI of PGF in CMV seropositive patients was 25% and in CMV seronegative patients was 14%. Of note, HHV6 viremia was detected in patients with PGF. HHV6 is not routinely monitored, however, making it difficult to establish a causative role. All patients had moderate PGF at diagnosis and 3 cases had worsening of cytopenias and met the criteria for severe PGF. To date, 3 PGF patients have died from EBV-PTLD, adenovirus infection or GVHD (developed after CMV treatment with liposomal cidofovir), 3 continue to suffer from PGF and 2 patients are alive with recovered good blood counts after eradication of CMV. Of the 3 patients with persistent PGF, one received a TCD boost with no response, and 2 continued to be treated for CMV viremia. A stem cell boost was indicated if pancytopenia persisted despite eradication of cause of the PGF. In this small series, there were not enough events to evaluate association between PGF and CD34 cell dose, CD3 cell dose or day 100 T-cell chimerism. Conclusions: In this homogenous population of patients with MDS who underwent TCD allogeneic HSCT, the incidence of PGF is about 20%. The most common cause was viral infection with predominance of CMV. Therefore, strategies to prevent CMV reactivation in patients undergoing allogeneic HSCT has the potential to reduce the risk of PGF and avoid the need for infusion of additional stem cells. Disclosures: No relevant conflicts of interest to declare.

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