Early Results of the Clinical Collection and Genetic Correction of Fanconi Complement Type A Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1283-1283
Author(s):  
Patrick Kelly ◽  
B. Balcik ◽  
K. Bohn ◽  
R. Mueller ◽  
I. Jurickova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Cell Recovery ◽  
Hematopoietic Stem ◽  
Genetic Syndrome ◽  
Cd34 Cells ◽  
Transfer Study

Abstract Fanconi anemia (FA) is a genetic syndrome characterized by almost uniform development of aplastic anemia. Current therapies for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors have shown high morbidity and mortality in clinical trials. Genetic correction of FA HSC using viral vectors has been demonstrated in animal models. However, harvesting of sufficient CD34+ cells at the time that HSC therapy is clinically indicated is difficult due to the severe bone marrow hypoplasia that accompanies pancytopenia. We have opened two phase I clinical trials that seek to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). These studies are being conducted with approval by the FDA/NIH-RAC/Institutional IRB and monitored by an independent DSMB. To date, 4 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.3x106 CD34+ cells/kg (range 0.3–2.9x106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells will be challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1x105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected using the CliniMACS device. Despite collection before significant pancytopenia, an average of only 5x105 CD34+ cells/kg (range 1.5–10x105 CD34+ cells/kg) was purified from these 3 cases representing ~10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final products determined by real-time PCR analysis of CD34+-derived progenitors averaged 48% (range 40–62%). Equivalent efficiency of correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82–110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5–3.5x105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. One patient is now 6 months post re-infusion with no evidence of gene marking observed in her PB or BM. The second patient had detectable FAA vector sequences in her PB early post-infusion (+4 weeks) but had none detected +8 weeks. The data suggest that while gene transfer efficiency in the clinical setting has been significantly improved, collection and expansion (either in vitro or in vivo) of adequate number of HSC may be critical to the success of genetic correction attempts in FA.

Ex Vivo Expansion and Long-Term Hematopoietic Reconstitution Ability of Sorted CD34+CD59+ Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2324-2324
Author(s):  
Juan Xiao ◽  
Bing Han ◽  
Wanling Sun ◽  
Yuping Zhong ◽  
Yongji Wu
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Peripheral Blood Cell ◽  
Intravascular Hemolysis ◽  
Blood Cell Count ◽  
Normal Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT). Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo. Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. The long-term hematopoietic supporting ability of expanded CD34+CD59+ cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency(SCID) mice in vivo. Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P<0.01 ), they remained strong colony-forming capacity even after expansion ( no difference was noticed in CFCs or LTC-IC of PNH CD34+CD59+ cells before and after expansion, P>0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells. Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

Targeted Gene Modification In Hematopoietic Stem Cells: A Potential Treatment For Thalassemia and Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 434-434
Author(s):  
Andreas Reik ◽  
Kai-Hsin Chang ◽  
Sandra Stehling-Sun ◽  
Yuanyue Zhou ◽  
Gary K Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Target Gene ◽  
Ex Vivo ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for <20% of cases, and (ii) the allogeneic nature of the transplant involves the significant risk of graft vs host disease (GvHD). Elevated levels of fetal γ-globin proteins observed in a subset of individuals carrying β-thal and SCD mutations ameliorate the clinical picture or prevent the development of disease complications. Thus, strategies for the selective and persistent upregulation of γ-globin represent an attractive therapeutic approach. Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

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.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1137-1137
Author(s):  
Tong Wu ◽  
Hyeoung Joon Kim ◽  
Stephanie E. Sellers ◽  
Kristin E. Meade ◽  
Brian A. Agricola ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Colony Pcr ◽  
High Level

Abstract Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.

Absence of the Rho GTPase Activating Protein p190-B Enhances Long Term Hematopoietic Stem Cell Engraftment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 614-614 ◽  
Author(s):  
Haiming Xu ◽  
Hartmut Geiger ◽  
Kathleen Szczur ◽  
Deidra Deira ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Gtpase Activating Protein ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Serial Transplantation

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of p190-B in HSC/P engraftment. Since mice lacking p190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and p190-B−/− embryos. WT and p190-B−/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of p190-B−/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted p190-B−/− animals, since LinnegScaposKitpos (LSK) cells were still present in the BM of p190-B−/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16Ink4a (Ito et al, Nat Med 2006), the expression of p16Ink4a was examined during serial transplantation. While expression of p16Ink4a increased in WT HSC in primary and secondary recipients, p16Ink4a remained low in p190-B−/− HSC, which indicated that p190-B-deficiency represses the upregulation of p16Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of p190-B-mediated HSC functions. We next examined whether p190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and p190-B−/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in p190-B−/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received p190-B−/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, p190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, p190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define p190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.

Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4168-4168
Author(s):  
Satiro N. De Oliveira ◽  
Francesca Giannoni ◽  
Cinnamon Hardee ◽  
Arineh Sahaghian ◽  
Laurence J N Cooper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Nsg Mice ◽  
Novel Approach ◽  
B Lineage

Abstract Abstract 4168 Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures: No relevant conflicts of interest to declare.

Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1192-1192
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Takafumi Hiramoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

Small Molecule Combination Enhances CD34+/CD38- Cell Proliferation Via Inhibition of Differentiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 658-658
Author(s):  
Lan Wang ◽  
Xin Guan ◽  
Huihui Wang ◽  
Bin Shen ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Small Molecules ◽  
Cell Expansion ◽  
Target Genes ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Qpcr Analysis ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Hematopoietic stem cells (HSCs) have become increasingly attractive for the therapy of various hematological system disorders. The aim of this study is to identify approaches that promote the expansion of HSCs. We present here the identification of a combination of small molecules and cytokines that is effective in retaining high stemness of hematopoietic stem/progenitor cells while promoting cell proliferation by inhibiting differentiation. Firstly, five small-molecule candidates were screened for their individual effects on ex vivo expansion of human peripheral blood CD34+ cells in the presence of selected cytokines. The best compounds at their optimal concentrations were further analyzed in combination, to achieve maximum capacity for stimulating the CD34+CD38- cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. Additional cell and molecular assays were performed to confirm that the expanded CD34± cells are functionally normal in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment ofhuman cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through qPCR during the process of CD34±cell expansion. Following multiple rounds of screening, an optimal formula (named as "SVC cocktail") was obtained, which consisted of four cytokines (stem cell factor, flt-3 ligand, thrombopoietin and interleukin-6) and three small molecules (Stem Regenin 1, valproic acid and CAY10433). CD34+ cells cultured with SVC cocktail had a purity of 76.2%±7.5% and reached expansion folds of 27.9±4.3 for CD34+/CD38- HSCs on day 7. In contrast, CD34+ cells cultured with the cytokines alone displayed a purity of 27.4%±6.3% and expansion folds of 15.5±2.2 for CD34+/CD38- cells. The groups with small molecules only (plus DMSO, the vehicle), or with basal medium only, showed no surviving cells on day 4. Furthermore, cell cycle analysis indicated that the SVC cocktail-induced CD34+/CD38- cells stayed in a more quiescent state (G0/G1: 75.2%±3.6%; S: 9.2%±2.4%). On the other hand, the cells cultured without the three small molecules had active DNA synthesis (G0/G1: 56.0%±2.0%; S: 31.8%±3.2%), implicating a trend of enhanced cell differentiation in the cytokine alone group. RT-qPCR analysis further demonstrated that the expression of HSC stemness markers CD90, CD133, CD117, ALDH1, Bmi1, HoxB4, GATA-2, Runx1, and CXCR4 were elevated in the SVC cocktail-induced CD34+ cells, but dramatically reduced or barely detectable in the cytokine alone group. In addition, CFU assays for the SVC cocktail group vs the cytokine alone group demonstrated BFU-E of 54.0±4.6 vs 11.7±1.5, CFU-GM of 71.0±2.7 vs 8.3±2.5, CFU-GEMM of 40.7±3.8 vs 5.0±2.0 and CFU-Mk of 6.7±1.5 vs 0.7±0.6, respectively. For the in vivo engraftment in mouse bone marrow, human CD45 rate in the SVC cocktail group was much higher than in the cytokine alone group (21.1%±2.7% vs 0.5%±0.1%); similar group differences were also found in the CD34+ and CD34+CD38- rate (7.7%±1.4% vs 1.6%±1.2% and 6.8%±2.2% vs 1.6%±0.1% respectively), all at 8 weeks post transplantation. Moreover, qPCR analysis of Notch and Wnt signaling pathways for cultured cells on day 7 showed that the expression of Notch target genes (related to high activation of HSC property) was enhanced in the SVC cocktail group compare to the cytokine group (HES5: 9.2±2.3 vs 3.6±1.4 in arbitrary units; HEY1: 6.3±1.9 vs 2.6±1.2; HES1: 3.2±1.3 vs 1.3±0.4; Notch1: 1.4±0.3 vs 1.2±0.3), whereas the expression of Wnt target genes (related to activation of HSC differentiation) was greater in the cytokine alone group than in the SVC cocktail group (CCND1: 10.1±4.3 vs 1.2±0.8; LEF1: 4.3±0.6 vs 2.9±0.2; PPAR D: 3.4±0.3 vs 1.5±0.1; FZD2: 1.8±0.2 vs 1.0±0.1). Taken together, our results show that the new SVC cocktail is able to retain the characteristics of HSCs remarkably well, by enhancing their expansion while inhibiting their differentiation. Mechanistically, it appears that the three small molecules can effectively inhibit the cytokines' pro-differentiation effects on CD34+CD38- cells without affecting the cytokines' ability to stimulate cell proliferation. Disclosures Wang: Biopharmagen Corp.: Employment. Ren:Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

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