scholarly journals Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 74-86 ◽  
Author(s):  
Valentina Capo ◽  
Sara Penna ◽  
Ivan Merelli ◽  
Matteo Barcella ◽  
Serena Scala ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Progenitor Cells ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Source

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

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

scholarly journals What Is the Most Appropriate Source for Hematopoietic Stem Cell Transplantation? Peripheral Stem Cell/Bone Marrow/Cord Blood

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Itır Sirinoglu Demiriz ◽  
Emre Tekgunduz ◽  
Fevzi Altuntas
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Cell Transplantation ◽  
Hematopoietic Stem ◽  
Stem Cell Source ◽  
Cell Source ◽  
Selection Of

The introduction of peripheral stem cell (PSC) and cord blood (CB) as an alternative to bone marrow (BM) recently has caused important changes on hematopoietic stem cell transplantation (HSCT) practice. According to the CIBMTR data, there has been a significant decrease in the use of bone marrow and increase in the use of PSC and CB as the stem cell source for HSCT performed during 1997–2006 period for patients under the age of 20. On the other hand, the stem cell source in 70% of the HSCT procedures performed for patients over the age of 20 was PSC and the second most preferred stem cell source was bone marrow. CB usage is very limited for the adult population. Primary disease, stage, age, time and urgency of transplantation, HLA match between the patient and the donor, stem cell quantity, and the experience of the transplantation center are some of the associated factors for the selection of the appropriate stem cell source. Unfortunately, there is no prospective randomized study aimed to facilitate the selection of the correct source between CB, PSC, and BM. In this paper, we would like to emphasize the data on stem cell selection in light of the current knowledge for patient populations according to their age and primary disease.

scholarly journals Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
DN Haylock ◽  
LB To ◽  
TL Dowse ◽  
CA Juttner ◽  
PJ Simmons
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

Abstract Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

scholarly journals Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
DN Haylock ◽  
LB To ◽  
TL Dowse ◽  
CA Juttner ◽  
PJ Simmons
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

scholarly journals Hematopoietic Stem Cell–Based Gene Therapy for Acquired Immunodeficiency Syndrome: Efficient Transduction and Expression of RevM10 in Myeloid Cells In Vivo and In Vitro

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2283-2290 ◽  
Author(s):  
Lishan Su ◽  
Robert Lee ◽  
Mark Bonyhadi ◽  
Hajime Matsuzaki ◽  
Sean Forestell ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Gene Transduction ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Hiv 1

Abstract Gene delivery via the hematopoietic stem cell (HSC) offers an attractive means to introduce antiviral genes into both T cells and macrophages for acquired immunodeficiency syndrome (AIDS) gene therapy. An amphotropic retroviral vector encoding a bicistronic gene coexpressing RevM10 and the murine CD8α′ chain (lyt2) was developed to transduce HSC/progenitor cells. After transduction of CD34+ cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytometry was used to monitor the level of gene transduction and expression and to enrich RevM10-expressing cells by cell sorting without drug selection. Using this quantitative method, high levels of gene transduction and expression (around 20%) were achieved by high-speed centrifugation of CD34+ cells with the retroviral supernatant (spinoculation). After reconstitution of human bone marrow implanted in SCID mice (SCID-hu bone) with the transduced HSC/progenitor cells, a significant number of donor-derived CD14+ bone marrow cells were found to express the RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus–type 1 (HIV-1) isolate was greatly inhibited in the lyt2+/CD14+ cells differentiated from transduced CD34+ cells after the enrichment of lyt2+ population. Thus, the RevM10 gene did not appear to inhibit the differentiation of HSC/progneitor cells into monocytes/macrophages. The level of retrovirus-mediated RevM10 expression in monocytes/macrophages derived from transduced HSCs is sufficient to suppress HIV-1 replication.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

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

Sign in / Sign up

Export Citation Format

Share Document