scholarly journals RUNX1 Mutations Cause a Myeloid Differentiation Block Leading to the Formation of a Long Term Expanding CD34+/CD33+/CD45RA+/CD123+ Cell Population

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1979-1979
Author(s):  
Myléne Gerritsen ◽  
Esther Tijchon ◽  
Amit Mandoli ◽  
Joost H.A. Martens ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Population ◽  
Molecular Mechanisms ◽  
Myeloid Differentiation ◽  
Dominant Negative Mutant ◽  
Chip Sequencing ◽  
Cd34 Cells ◽  
Differentiation Block ◽  
Homogenous Cell Population

Abstract RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant by in vitro and in vivo experiments and RNA- and ChIP-sequencing approaches. Overexpression of the RUNX1 mutant S291fs300X in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) while the CFU-GM colonies showed enhanced replating capacity compared to control (>3 times). It appeared that the replating potential was restricted to CD14-/CD15- progenitor cells. Long-term suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 S291fs300X CB CD34+ cells provided a rather homogenous cell population after 10 weeks of culture. These cells are growth factor dependent and are phenotypically defined by CD34+/CD38+/CD33+/IL1-RAP+/CD45RA+/CD123+ resembling a GMP phenotype which can be propagated for approximately 20 weeks in suspension. Comparable results were obtained with normal bone marrow CD34+cells transduced with the RUNX1 S291fs300X. Karyotype analyses demonstrated no abnormalities while integration site analysis showed a variety of different integration sites and differences between individual samples, suggesting that the myeloid differentiation block is related to the RUNX1 S291fs300X mutation.Long-term MS5 stromal co-cultures of transduced RUNX1 S291fs300X CB CD34+ cells showed after 8-10 weeks a rather homogenous cell population with limited potential to expand and localized under the stromal layer. This cell population is phenotypically defined by CD34+/CD38-. The interactions with the stroma appear to prevent proliferation but retain quiescence, indicating that sufficient niche-cell interactions might be crucial for transformation. NSG mice experiments are performed to test the reproducibility of these findings in vivo. Q-PCR studies demonstrated reduced expression of C/EBPα in RUNX1 S291fs300X CB CD34+ cells, one of the key targets in myeloid differentiation. Therefore, week 10 RUNX1 S291fs300X CB CD34+ cells were transduced with a retroviral C/EBPα overexpression vector. The re-expression of C/EBPα resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression. RNA- and ChIP-sequencing data revealed a decreased expression of crucial RUNX1 target genes including C/EBPα and Cited2 and also a retained binding of mutant RUNX1 on these loci in conjunction with a decrease of H3K27ac. Further research into the molecular mechanisms by which this RUNX1 S291fs300Xderegulates gene-expression is in progress. Our results implicate that overexpression of RUNX1 S291fs300X mutant leads to impaired erythroid differentiation and a strong differentiation block of the myeloid lineage resulting in the expansion and maintenance of a GMP-like cell population. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4102-4109 ◽  
Author(s):  
CI Civin ◽  
G Almeida-Porada ◽  
MJ Lee ◽  
J Olweus ◽  
LW Terstappen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Fetal Sheep ◽  
Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

Stromal Derived Factor-1–Induced Chemokinesis of Cord Blood CD34+ Cells (Long-Term Culture-Initiating Cells) Through Endothelial Cells Is Mediated by E-Selectin

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4011-4019 ◽  
Author(s):  
Afzal J. Naiyer ◽  
Deog-Yeon Jo ◽  
Jongcheol Ahn ◽  
Robert Mohle ◽  
Mario Peichev ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecules ◽  
Constitutive Expression ◽  
Transendothelial Migration ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Migration System ◽  
Cd34 Cells

Abstract Homing of hematopoietic stem cells to the bone marrow (BM) involves sequential interaction with adhesion molecules expressed on BM endothelium (BMEC) and chemokine stromal derived factor-1 (SDF-1). However, the mechanism whereby adhesion molecules regulate the SDF-1–induced transendothelial migration process is not known. E-selectin is an endothelial-specific selectin that is constitutively expressed by the BMEC in vivo. Hence, we hypothesized that E-selectin may mediate SDF-1–induced transendothelial migration of CD34+ cells. We show that CD34+ cells express both E-selectin ligand and fucosyltransferase-VII (FucT-VII). Soluble E-selectin–IgG chimera binds avidly to 75% ± 10% of CD34+ cells composed mostly of progenitors and cells with long-term culture-initiating cell (LTC-IC) potential. To assess the functional capacity of E-selectin to mediate CD34+ cell migration in a transendothelial migration system, CD34+ cells were placed on transwell plates coated with interleukin-1β–activated BMEC. In the absence of SDF-1, there was spontaneous migration of 7.0% ± 1.4% of CD34+ cells and 14.1% ± 2.2% of LTC-IC. SDF-1 induced migration of an additional 23.0% ± 4.4% of CD34+cells and 17.6% ± 3.6% of LTC-IC. Blocking MoAb to E-selectin inhibited SDF-1–induced migration of CD34+ cells by 42.0% ± 2.5% and LTC-IC by 90.9% ± 16.6%. To define the mechanism of constitutive expression of E-selectin by the BMEC in vivo, we have found that vascular endothelial growth factor (VEGF165) induces E-selectin expression by cultured endothelial cells. VEGF-stimulated endothelial cells support transendothelial migration of CD34+ cells that could be blocked by MoAb to E-selectin. These results suggest that trafficking of subsets of CD34+ cells with LTC-IC potential is determined in part by sequential interactions with E-selectin and SDF-1.

AMD3100-Mobilized CD34+ Cells Are Phenotypically Different and Better Targets for Retroviral Transduction Than G-CSF-Mobilized CD34+ Cells in Rhesus Macaques.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2686-2686
Author(s):  
Andre Larochelle ◽  
Allen Krouse ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Genetic Manipulation ◽  
Rhesus Macaques ◽  
Post Transplantation ◽  
Alternative Source ◽  
Cd34 Cells

Abstract AMD3100 (AMD) has recently been shown to rapidly mobilize primitive hematopoietic cells in mice and humans, but little is known about the properties of cells mobilized with this agent. We initiated a study to determine retroviral (RV) in vivo gene marking efficiency in AMD-mobilized CD34+ cells in rhesus macaques. CD34+ cells collected 3 hours after administration of AMD to 2 animals were transduced using RV vectors containing the NeoR gene. Animals were irradiated and cells reinfused immediately after transduction. By molecular analysis, the levels of PB MNC and granulocyte NeoR gene marking at steady-state (up to 12 months post-transplantation) was 1–2% in animal RC909 and 30–40% in RQ2851. In two additional rhesus macaques, CD34+ cells were collected from steady-state BM and from the PB after mobilization with AMD or G-CSF (G). The two PB populations from each animal were transduced with one of two distinguishable NeoR vectors and simultaneously reinfused into irradiated animals. In animal RQ3590, 2% in vivo gene marking at steady-state (up to 4 months post-transplantation) was derived from AMD-mobilized cells compared to 0.05% from the G-mobilized fraction. Animal RQ3636 showed 10% in vivo marking from the AMD-mobilized fraction and no detectable marking from the G-mobilized cells. We also compared phenotypic and functional characteristics of CD34+ cells from BM, AMD-PB and G-PB. An average of 31% of the AMD-mobilized cells were in the Go phase of the cell cycle, compared to 79% of G-mobilized cells (p=0.02), and 45% for the BM fraction (p=0.24). In contrast, 64% AMD-mobilized cells were in G1 compared to 17% of G-mobilized cells (p=0.03) and 44% for the BM fraction (p=0.15). Flow cytometry showed CXCR4 expression on 59% AMD-mobilized cells, in comparison to 11% G-mobilized cells (p=0.02) and 22% BM cells (p=0.07). Similar results were obtained when comparing VLA-4 expression. The increased expression of CXCR4 on AMD-mobilized CD34+ cells correlated with their increased ability to migrate towards SDF-1α in vitro (45%) compared to G-mobilized cells (8%, p=0.01) and BM cells (17%, p=0.08). Our data indicate efficient long-term in vivo gene marking in the rhesus macaque model, validating the ability of AMD to induce mobilization of true long-term repopulating HSCs. AMD-mobilized PB HSCs represent an alternative source of HSCs amenable to genetic manipulation with integrating RV vectors, with potential applications in gene therapy approaches for patients with sickle cell anemia; documented complications have precluded mobilization using G or G/SCF in these patients. Also, cell cycle status and surface phenotype of AMD-mobilized CD34+ cells are more comparable to steady-state BM cells than G-mobilized PB HSCs. AMD-mobilized CD34+ cells are more actively cycling than G-mobilized CD34+ cells, correlating with the increased efficiency of replication-dependent retrovirus-mediated gene transduction. The increased expression of the adhesion receptors CXCR4 and VLA-4 on primitive AMD-mobilized cells compared to G-mobilized cells suggests fundamental differences in the mechanisms of AMD-mediated and cytokine-mediated stem cell mobilization.

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.

Impaired GvL Potential of Natural Killer Cells Early Reconstituting Following Haploidentical HSCT.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3223-3223
Author(s):  
Luca Vago ◽  
Elisabetta Zino ◽  
Simona Di Terlizzi ◽  
Barbara Forno ◽  
Maria T. Lupo Stanghellini ◽  
...  
Keyword(s):  
Nk Cells ◽  
Low Dose ◽  
Functional Differences ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Long Term Follow Up ◽  
Leukemia Relapse

Abstract Alloreactive NK cells have been suggested to be important functional players in GvL activity after haploidentical HSCT for high risk leukemia. In this study we have characterized NK cells differentiating from purified haploidentical CD34+ cells after transplantation into 16 patients who did (n=8) or did not (n=8) suffer acute leukemia relapse in a long term follow-up (median 208 days). The incidence of relapse in these patients was not correlated with the presence (n=9) or absence (n=7) of predicted donor NK alloreactivity (p=0.94). NK cells in the first month after transplantation were, regardless of the occurence of relapse, NKG2A+ (>95%) and KIR− (13%), thus resembling CD56bright NK cells from healthy donors. However, in contrast to mature CD56bright cells, the patients’ NK cells expressed heterogeneous intensities of CD56, were only partly positive for the lymph node homing markers CD62L and CCR7, and expressed a higher amount of Fcγ receptor III (CD16). Importantly, in contrast to mature CD56bright cells, which constitrutively express the high affinity αβγ IL-2 receptor, thus releasing γ-IFN in response to low dose IL2, the patients’ NK cells lacked IL-R α (CD25) and did not release cytokines in response to low-dose IL2, nor, most importantly, when challenged with leukemic blasts. γ-IFN release induced by leukemic blasts could be restored by inhibition of NKG2A while cytotoxicity, which was consistently lower as compared to that of mature CD56+ cells, could not. Our data suggest that NK cells differentiating in patients from CD34+ progenitors after haploidentical HSCT have important phenotipical and functional differences from both subsets of mature NK cells, accounting for an impaired in vivo GvL potential.

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&lt;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&gt;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&gt;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&gt;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.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

Uncovering Haematopoietic System Dynamics and Single Multipotent Progenitors Activity In Vivo In Humans by Retroviral Tagging.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2611-2611
Author(s):  
Luca Biasco ◽  
Cristina Baricordi ◽  
Stefania Merella ◽  
Cynthia Bartholomae ◽  
Alessandro Ambrosi ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Human Model ◽  
The Real ◽  
Multipotent Progenitors ◽  
Cd34 Cells ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Abstract Abstract 2611 The long-standing model of human haematopoiesis postulates that myeloid and lymphoid lineages branch separately at very early stages, producing myeloid or erythroid cells and T or B cells, respectively. Conversely, a revised scheme of haematopoietic hierarchy was recently proposed, in which myeloid cells represent a prototype of blood cells, while erythroid, T and B cells are specialized cell types. The validity of these models has been mainly tested in vivo in the mouse, and in vitro through clonal assays on human haemopoietic stem cells (HSC). However, a clear definitive elucidation of the real nature of human haemopoiesis should ideally involve the ability to track the dynamics, survival and differentiation potential of haemopoietic progenitor clones for a long period of time directly in vivo in humans. Upon retroviral gene transfer, transduced cells are univocally tagged by vector insertions allowing them to be distinguished and tracked in vivo by integration profiling. We previously showed that gene therapy (GT) for adenosine deaminase (ADA) deficient SCID based on infusion of transduced CD34+ cells and reduced intensity conditioning, resulted in full multilineage engraftment, in the absence of aberrant expansions. Therefore, long-term studies in these patients provide a unique human model to study in depth haemopoietic clonal dynamics by retroviral tagging. For this reason, we performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cell subpopulations) cells in 4 patients 3–6 years after GT, retrieving to date 1055 and 1999 insertions from BM and PB cell lineages respectively. We could shape the insertional landscape of each lineage through a tri-factorial analysis based on the number of integrations retrieved, the percentage of vector positive cells and the number of insertion shared with other lineages. We were able to uncover the effects of selective advantages of gene-corrected cells in periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of gene-corrected progenitors. On the other hand, PB samples carried in general a higher frequency of vector positive cells, with the exception of PB CD15+ cells showing insertional landscapes very similar to the one of BM lineages. Interestingly, the detection of exclusively shared myeloid-T\B or myeloid-erythroid integrants may be supportive of a myeloid-based haemopoiesis model. We also uncovered “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages, stably tagging active long-term multipotent progenitors overtime. Strikingly, one of these progenitor clones carried an insertion inside one of the two fragile sites of MLLT3 gene, involved by translocation events in mixed lineage leukemia. We were able to track this and another integrant (downstream the LRRC30 gene) by specific PCRs, confirming the multilineage contribution to haematopoiesis of the relative progenitor clones and their fluctuating lineage outputs over 4 years, without showing aberrant expansions. We also retrieved 170 and 174 integrations from 4 T cell subtypes (Naive, TEMRA, Central and Effector memory) in two patients under PBL-GT and HSC-GT respectively. We found evidences that single naive T cell clones may survive in patients for up to 10 years after last infusion while maintaining their differentiation capacity into different T cell subpopulations. Interestingly, a cluster of 4 insertions (one of them shared among all T cell subtypes) was found in proximity of the interferon regulatory factor 2 binding protein 2 (IRF2BP2) gene in naive T cells from PBL-GT patient, thus suggesting an influence of transcriptional activity of this region on selective advantage of gene-corrected lymphocytes. In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans. The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients. Disclosures: No relevant conflicts of interest to declare.

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1920-1920
Author(s):  
Santosh Saraf ◽  
Hiroto Araki ◽  
Benjamin Petro ◽  
Kazumi G Yoshinaga ◽  
Simona Taioli ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Transcript Levels ◽  
Epigenetic Modifiers ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Abstract 1920 Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2'-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures: No relevant conflicts of interest to declare.

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