In Vitro and In Vivo Selection of Genetically Modified Cells Using shRNA Against HPRT and Treatment with 6-thioguanine.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2590-2590
Author(s):  
Christopher C. Porter ◽  
James DeGregori
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Genetically Modified ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Selection Of

Abstract Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Ex Vivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies

2018 ◽  
Vol 26 (2) ◽  
pp. 480-495 ◽  
Author(s):  
Kanit Bhukhai ◽  
Edouard de Dreuzy ◽  
Marie Giorgi ◽  
Charlotte Colomb ◽  
Olivier Negre ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Selection Of

scholarly journals Maturation of hematopoietic stem cells from prehematopoietic stem cells is accompanied by up-regulation of PD-L1

2017 ◽  
Vol 215 (2) ◽  
pp. 645-659 ◽  
Author(s):  
Joanna Tober ◽  
Marijke M.W. Maijenburg ◽  
Yan Li ◽  
Long Gao ◽  
Brandon K. Hadland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Responses ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Programmed Death

Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo–matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.

scholarly journals Cell and Gene Therapy for Anemia: Hematopoietic Stem Cells and Gene Editing

2021 ◽  
Vol 22 (12) ◽  
pp. 6275
Author(s):  
Dito Anurogo ◽  
Nova Yuli Prasetyo Budi ◽  
Mai-Huong Thi Ngo ◽  
Yen-Hua Huang ◽  
Jeanne Adiwinata Pawitan
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Ex Vivo ◽  
Risk Mitigation ◽  
Future Research ◽  
Hematopoietic Stem ◽  
Cell And Gene Therapy

Hereditary anemia has various manifestations, such as sickle cell disease (SCD), Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency (G6PDD), and thalassemia. The available management strategies for these disorders are still unsatisfactory and do not eliminate the main causes. As genetic aberrations are the main causes of all forms of hereditary anemia, the optimal approach involves repairing the defective gene, possibly through the transplantation of normal hematopoietic stem cells (HSCs) from a normal matching donor or through gene therapy approaches (either in vivo or ex vivo) to correct the patient’s HSCs. To clearly illustrate the importance of cell and gene therapy in hereditary anemia, this paper provides a review of the genetic aberration, epidemiology, clinical features, current management, and cell and gene therapy endeavors related to SCD, thalassemia, Fanconi anemia, and G6PDD. Moreover, we expound the future research direction of HSC derivation from induced pluripotent stem cells (iPSCs), strategies to edit HSCs, gene therapy risk mitigation, and their clinical perspectives. In conclusion, gene-corrected hematopoietic stem cell transplantation has promising outcomes for SCD, Fanconi anemia, and thalassemia, and it may overcome the limitation of the source of allogenic bone marrow transplantation.

The spleen is a major site of megakaryopoiesis following transplantation of murine hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3975-3982 ◽  
Author(s):  
William B. Slayton ◽  
Ann Georgelas ◽  
L. Jeanne Pierce ◽  
Kojo S. Elenitoba-Johnson ◽  
S. Scott Perry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Glucose Phosphate ◽  
Stem Cell Pool

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rholow hematopoietic stem cells and Rhohigh progenitors. Rholow stem cells permanently engraft all lineages, whereas Rhohighprogenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rhohigh cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rhohigh progenitors produced more megakaryocytes in vitro than Rholow stem cells did. To study the activity of the Rholow and Rhohighsubsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rholow stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rhohigh progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donor-derived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rholowstem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoiesis following engraftment of transplanted stem cells in irradiated mice.

Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors

Blood ◽  
2014 ◽  
Vol 123 (8) ◽  
pp. 1167-1177 ◽  
Author(s):  
Patricia Benveniste ◽  
Pablo Serra ◽  
Dzana Dervovic ◽  
Elaine Herer ◽  
Gisele Knowles ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Immune Reconstitution ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Key Points

Key Points Notch signals expand human HSC (CD90low) cells in vitro and delay the expansion of CD45RAint and CD45RAhi cells in vitro. HSCs expanded in vitro are equal to ex vivo CD90low cells in immune reconstitution.

Clonal Analysis of Hematopoietic Stem Cells after Serial Transplantation of Animals Receiving MGMTP140K Gene Transduced Cells Following Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1182-1182
Author(s):  
Stephanie Laufs ◽  
Ursula Sorg ◽  
Veronika Kleff ◽  
Laila Gao ◽  
Michael Flasshove ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Serial Transplantation ◽  
Selection Of

Abstract Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells has been shown to protect hematopoiesis from the toxic side effects of O6-guanine alkylating drugs such as BCNU, ACNU or temozolomide (TMZ). In addition, MGMT gene transfer allows efficient in vivo selection of transduced hematopoietic stem cells and enrichment of genetically corrected cells in the context of gene therapy for monogenetic diseases. We here have analysed the long-term effect of MGMT gene transfer on the hematopoietic stem cell compartment using an in vivo murine transplantation/gene therapy model and a retroviral vector carrying the gene for MGMTP140K, a mutant resistant to the wtMGMT-specific inhibitor O6-benzylguanine (BG). Serial transplants were performed and primary, secondary as well as tertiary recipients were treated with combined BG/ACNU, BG/BCNU or BG/TMZ chemotherapy at doses myeloablative in non-MGMT-protected hematopoiesis. Serial transplantation was performed with 1.8 – 3.0 x 106 mononuclear bone marrow cells and 2 to 3 animals were transplanted per primary or secondary animal. While initial gene transfer efficiency was low (1–5% of cells engrafted at week four) chemotherapy resulted in efficient selection of transduced cells in primary animals (70–90% transgene expression in peripheral blood). Secondary and tertiary recipients showed 40–80% transgene expression even before CTX. Efficient stem cell engraftment and protection from CTX was demonstrated in > 90% of secondary animals, while tertiary recipients clearly demonstrated compromised engraftment and a substantial number of animals did not survive CTX treatment. Retroviral vector integration site analysis to study the clonality of hematopoiesis of stem cells by ligation mediated PCR (LM-PCR) was performed in the serially transplanted mice. In three mice of the secondary transplantation cohort we detected 3, 0, and 6 clones, respectively. In three mice of the tertiary transplantation cohort 7, 2, and 2 clones, respectively, were found. Thus, an exhaustion of transduced hematopoiesis following regenerative stress by high dose chemotherapy was not evident. Of the total 20 detected clones one could not be mapped to the mouse genome, while the others could be blasted against the mouse genome (assembly 2004, http://genome.ucsc.edu/; >99.5% identity). It turned out that 5 of 8 integrations landed in RefSeq in the tertiary transplantation cohort, while 3 of 8 integrations occurred in RefSeq genes in the secondary transplantation cohort. This suggests that clones profit from the transcription machinery of their integration site. Thus, our LM-PCR results indicate that the multiclonality of hematopoiesis is conserved after serial transplants which may be considered a safety feature for drug-resistance gene therapy. Furthermore, vector integration in highly resistant stem cells is favored in actively transcribed genomic regions.

Ex Vivo Treatment of Human Cord Blood with dmPGE2 Can Safely Increase the Potential for Hematopoietic Engraftment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1190-1190
Author(s):  
Trista E. North ◽  
Wolfram Goessling ◽  
Myriam Armant ◽  
Grace S. Kao ◽  
Leslie E. Silberstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Relative Distribution ◽  
Human Cord Blood ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are commonly used in transplantation therapy to rescue the hematopoietic and immune systems following systemic chemotherapy or irradiation. However, some patients receive inadequate numbers of HSCs and this often results in delayed reconstitution of hematopoiesis and immune function and associated toxicities. We previously demonstrated that a stabilized derivative of prostaglandin (PG) E2 increases vertebrate HSCs both in vivo and in vitro. 16,16-dimethyl PGE2 (dmPGE2) significantly increased HSCs during zebrafish embryogenesis and in the adult marrow following injury. Incubation of murine embryonic stem cells with dmPGE2 during embryoid body differentiation resulted in a dose-dependent increase in hematopoietic colonies, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Finally, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in engrafting cells in a limiting dilution competitive repopulation assay. No negative effects on serial transplantability of HSCs were observed in these animal models. To investigate the therapeutic potential of PGE2 for the amplification of blood stem cells, we exposed human cord blood (hCB) cells to dmPGE2 in vitro and measured the effects on stem and progenitor populations both in vitro and in vivo. Red cell depleted umbilical cord blood specimens, cryopreserved for clinical use, were thawed and divided for parallel processing. Ex vivo treatment of hCB cells for 1 hour with dmPGE2 in dextran/albumin had no negative impact on absolute cell count or the viability and relative distribution of both CD45 and CD34 positive cells compared to vehicle treated control hCB cells. Significantly, hCB treated with dmPGE2 produced enhanced numbers of GM and GEMM colonies in methylcellose CFU-C assays compared to controls. Human CB cells treated ex vivo with dmPGE2 for 1 hour and transplanted at a dose of 20 million live CD45+ cells per recipient were capable of repopulating NOD/SCID mice after sublethal irradiation. In comparative studies at 6 weeks post transplantation, human CD34+ and CD45+ cells could be detected in the marrow (>2%) of dmPGE2 treated (4/8) and control treated (1/6) recipients. Long-term and competitive transplantation experiments to assess the effect of dmPGE2 treatment on functional HSCs are currently in progress. Our data suggests that treatment of human cord blood products with dmPGE2 will be both safe and effective in achieving expansion of hematopoietic stem cells for transplantation in the clinical setting. TE North and W Goessling contributed equally to this work.

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