Long-Term Coculture of Adult Human Healthy Donor CD34+ Cells Shows Elevation of Telomeres in a Subgroup of Donors Independent of Age, Stem Cell Function and Telomerase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1695-1695
Author(s):  
Katja C. Weisel ◽  
Kaida Wu ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Telomere Length ◽  
Healthy Donor ◽  
Progenitor Cell ◽  
Telomerase Activity ◽  
Suspension Cells ◽  
Telomere Shortening ◽  
Cd34 Cells

Abstract Long-term cytokine-supplemented or stromal cocultures of human CD34+ cells, particularly from cord blood (CB), show expansion of hematopoietic progenitors and stem cells. Ultimately, however cultures decline and terminally differentiate. Despite upregulation of telomerase activity in proliferating primitive hematopoietic cells, telomere shortening has generally been reported in long-term cultures of CB, bone marrow or G-CSF mobilized peripheral blood CD34+ cells. In earlier reports, we described a long-term culture of hematopoietic stem cells on a murine OP9 bone marrow stroma cell line transfected with an adenovector expressing thrombopoietin, which allowed an extensive proliferation and self-renewal of CB CD34+ cells for 4–5 months with sustained elevation of telomerase activity and without concomitant significant telomere shortening (Blood, 2004). Here, we evaluated adult healthy donor peripheral blood (PB) CD34+ cells in the same OP9/Tpo coculture system. To determine progenitor and stem cell production, standard CFC and 2ndry cobblestone area-forming cell assays (CAFC assayed at 5 weeks on MS5 stroma) were undertaken weekly with suspension cells. In addition telomere length was measured by telomere restriction fragment (TRF) assay, and telomerase activity by TRAP assay on input CD34+ cells, and weekly on culture suspension cells. Maximum total cell, CFC and CAFC production was seen in the first 4 weeks with up to 80-fold expansion in cell count, up to 4-fold expansion in CFC and up to 13-fold expansion in CAFC. Thereafter a continuous decrease in production of cells, CFC 2ndry CAFC was observed and cultures terminated at week 8. Mean telomere length of input PB CD34+ cells was 9,5 ± 0,5 kbp. After 4 weeks in culture, telomere length remained stable (9,6 ± 0,5 kbp). In 3/6 cultures terminated, cultures showed only a slightly decrease of telomere length compared to the input population (0,55 ± 0,1 kbp loss). However, in 2/6 cultures we could demonstrate an elevation of telomeres (+ 0,3 kbp) independent of a rapid loss of telomerase activity in all cultures during the culture period. Furthermore, the elevation of telomeres did not correlate with an enhanced stem/progenitor cell activity. These data confirm earlier results of granulocyte telomere change in myeloma patients following chemotherapy and tandem transplantation, where 154/193 patients showed an expected loss of telomeres during the treatment period, whereas 39/193 patients had an unexpected elevation of telomeres. We could now show in healthy donors that this phenomenon is independent of bone marrow stress due to chemotherapeutic treatment. In conclusion, we could show that the stromal coculture system with OP9/Tpo is highly effective in stem/progenitor cell expansion not only in CB but also in PB CD34+ cells. It is the first culture system, which allows expansion of hematpoietic cells without significant telomere erosion. We furthermore describe for the first time an age-independent healthy donor population which shows telomerase-independent, significant telomere elevation. Further studies have to demonstrate, if this phenomenon is potentially linked to a higher susceptibility for cancer disease.

scholarly journals Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-18
Author(s):  
Wenxue Ma ◽  
Larissa Balaian ◽  
Phoebe Mondala ◽  
Yudou He ◽  
Cayla Mason ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Telomere Length ◽  
Mouse Models ◽  
Telomerase Activity ◽  
Rna Seq ◽  
Publicly Traded ◽  
Cd34 Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4440-4448 ◽  
Author(s):  
Loretta Gammaitoni ◽  
Katja C. Weisel ◽  
Monica Gunetti ◽  
Kai-Da Wu ◽  
Stefania Bruno ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Telomere Length ◽  
Severe Combined Immunodeficiency ◽  
Telomerase Activity ◽  
Clinical Value ◽  
Telomere Shortening ◽  
Cd34 Cells

Abstract Telomerase activity, telomere length, stem/progenitor cell production, and function of CD34+ cells from cord blood (CB), bone marrow, and mobilized peripheral blood were evaluated in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34+ cells reisolated at monthly intervals for passage. Continuous expansion of stem cells as measured by in vitro cobblestone area and secondary colony-forming assays was noted for 18 to 20 weeks and by severe combined immunodeficiency (SCID)-repopulating cells (SRCs), capable of repopulating and serially passage in nonobese diabetic/SCID mice, for 16 weeks. Despite this extensive proliferation, telomere length initially increased and only at late stages of culture was evidence of telomere shortening noted. This telomere stabilization correlated with maintenance of high levels of telomerase activity in the CD34+ cell population for prolonged periods of culture. Cytokine-stimulated cultures of adult CD34+ cells showed CD34+ and SRC expansion (6-fold) for only 3 to 4 weeks with telomere shortening and low levels of telomerase. There is clearly a clinical value for a system that provides extensive stem cell expansion without concomitant telomere erosion. (Blood. 2004;103:4440-4448)

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2617-2617
Author(s):  
Fumio Arai ◽  
Kentaro Hosokawa ◽  
Yumiko Nojima ◽  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2617 Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs. On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004). In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT. These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3754-3761 ◽  
Author(s):  
R Haas ◽  
B Witt ◽  
R Mohle ◽  
H Goldschmidt ◽  
S Hohaus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Lymphoblastic Leukemia ◽  
Peripheral Blood Progenitor Cell ◽  
Myelogenous Leukemia ◽  
High Dose ◽  
Platelet Recovery ◽  
Cd34 Cells

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non- Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.

Telomere Length of Hematopoietic Cells in Long-Term Post-Autograft Survivors: No Evidence of Accelerated Replicative Cell Senescence Despite the Persistent Reduction of Both Committed and Immature Progenitor Cell Compartments.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4123-4123
Author(s):  
Alberto Rocci ◽  
Irene Ricca ◽  
Chiara Della Casa ◽  
Paolo Longoni ◽  
Mara Compagno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Progenitor Cell ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Replicative Stress

Abstract Telomere length is considered a valuable replicative capacity predictor of human hematopoietic stem cells. Indeed, a progressive telomere shortening affects hematopoietic cells upon in vitro expansion. However, less is known on the dynamics of telomere shortening in vivo following a non-physiological replicative stress. Aim of this study was to investigate markers for cellular senescence of hematopoietic cells exposed to replicative stress induced by bone marrow reconstitution following stem cell autograft. Thus, both telomere length and in vitro functional characteristics of bone marrow (BM) and peripheral blood (PB) were evaluated at long-term in subjects who had received intensive chemotherapy and autograft. Thirty-two adults with a previous diagnosis of lymphoma were examined, at a median time of 73 months (range 42–125) since autograft. They all had received a high-dose sequential chemotherapy treatment followed by peripheral blood progenitor cell (PBPC) autograft. There were 20 male and 12 female (median age at autograft: 40 yrs., range 21–60). A Southern blot procedure using a chemiluminescence-based assay was employed to determine telomere length on samples from grafted PBPC as well as on BM and PB samples obtained at long-term during follow-up. These latter samples were also studied for their in vitro growth characteristics, assessed by short and long-term culture assays. In all cases, autograft had been performed with large quantities of hematopoietic stem cells (median autografted CD34+ve cells/kg: 9.8 x 106, range 2–24), allowing a rapid and stable hematologic reconstitution. Telomere length was found slightly shorter in BM mononuclear cells from samples taken at follow-up compared to samples from grafted material (median telomere length: 6,895 bp vs 7,073 bp, respectively; p=ns). No marked differences were observed in telomere evaluation between BM and PB cells. No significant differences were observed as well when PB telomere length of follow-up samples was compared with telomere length of PB from age-related normal subjects. BM and PB samples were then assessed for their in vitro growth characteristics. Committed and stromal progenitors were grown from all samples in good though variable quantities. However, as compared to normal controls, a statistically significant reduction of marrow-derived hematopoietic progenitors (CFU-GM - BFU-E - CFU-Mix) as well as stromal progenitors (CFU-F) was observed. Additionally, the more immature LTC-IC progenitor cell compartment was dramatically reduced, both in BM and PB samples. The results indicate that: i. the proliferative stress induced by intensive chemotherapy and post-graft hematopoietic reconstitution does not imply marked telomere loss in BM and PB cells at long-term, provided that large quantities of PBPC are used for autograft; ii. stem cells present in the graft or surviving after high-dose therapy are capable of reconstituting a sufficiently adequate hematopoiesis although the committed progenitor cell compartment and even more the immature LTC-IC progenitors are persistently reduced even at up to 10 years since autograft.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3279-3279
Author(s):  
Samantha Scaramuzza ◽  
Sara Trifari ◽  
Francesco Marangoni ◽  
Silvana Martino ◽  
Ayse Metin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Efficacy And Safety ◽  
Wiskott Aldrich Syndrome ◽  
Cd34 Cells ◽  
Was Gene

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

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

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

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

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

2020 ◽  
Vol 66 (3) ◽  
pp. 265-273
Author(s):  
O.V. Vysotskaya ◽  
A.I. Glukhov ◽  
Yu.P. Semochkina ◽  
S.A. Gordeev ◽  
E.Yu. Moskaleva
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cell Lines ◽  
Telomerase Activity ◽  
Mouse Bone Marrow ◽  
Fibrosarcoma Cell

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

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