Long-term Culture-initiating Cell Activity Research Articles

Architectural and Functional Heterogeneity of Hematopoietic Stem/Progenitor Cells in Non-Del(5q) Myelodysplastic Syndromes

IntroductionMyelodysplastic syndromes (MDS) are clinically diverse malignant disorders of aging with a propensity to evolve to acute myeloid leukemia (AML) or bone marrow failure. In early MDS, whole genome sequencing identified mutations distributed in few clones. Evidence have been provided for the existence of a MDS-initiating cell in cases harboring a 5q deletion. However, the clonal heterogeneity and its consequences on the phenotypic diversity of non-del(5q) MDS is little documented. Here we focused on studying the hierarchical organization and the functionality of clones defined by molecular profiling. The clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment was investigated and dominant clones were examined as MDS-initiating cells.Material and methodsBone marrow (BM) samples were obtained from 20 patients with non-del(5q) MDS enrolled in the national Programme Hospitalier de Recherche Clinique MDS-04 after informed consent in accordance with ethics committee guidelines. BM samples,cytaphereses from age-matched healthy individuals and cord bloods were used as controls. Targeted NGS of a selected panel of 39 genes was used to define the mutational landscape on BM mononuclear cells (MNC). To study the clonal architecture at the HSPC level, single CD34+CD38- cells were seeded in 96-well plates coated with MS-5 stromal cells and cultured in H5100 MyeloCult medium (StemCell Technologies, Vancouver, Canada) with cytokines for six weeks. For long-term culture-initiating cell (LTC-IC) assays, CD34+ progenitors were cultured for six weeks on MS-5-coated plates without cytokines and then tested for colony-forming cells. For clonogenic assays, CD34+CD38- cells were seeded in methylcellulose for two weeks. All animal experimentations were performed in NSG mice.ResultsIn the 20 cases of non-del(5q) MDS, genomic lesions were traced down to single CD34+CD38- HSPC-derived colonies. Clonal organization was mostly linear in 13/17 patients and branched in 4 cases with retention of a dominant subclone. The clone detected in LTC-IC compartment and that reconstituted short-term human hematopoiesis in xenotransplantation models was usually the dominant clone, which gave rise to the myeloid and to a lesser extent to the lymphoid lineage. Other mutations not detected in LTC-IC can appear in CD34+CD38- compartment or at the level of lineage-committed progenitors. The pattern of mutations may differ between common myeloid (CMP), granulo-monocytic (GMP) and megakaryocytic-erythroid (MEP) progenitors. For instance, a major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knockdown by shRNA in normal CD34+ progenitors impaired their granulocytic and erythroid differentiation. By contrast, a STAG2 gene mutation, not detected in CMP or MEP, amplified in a GMP, which drove the transformation to AML.ConclusionIn the present study, we characterized the first genetic hits that initiate disease in a dominant clone of the CD34+CD38- HSPC compartment, which exhibits LTC-IC activity and reconstitutes human short-term hematopoiesis in NSG mice. The genetic heterogeneity in non-del(5q) MDS arises within the HSPC compartment and in lineage-committed progenitors which ultimately support the transformation into AML. The clonal architecture of HSPC compartment and mutations selection along differentiation contribute to the phenotype of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation, and may guide the search for novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Effective Development of Hematopoietic Niche Cells From hESCs: hESC-Derived Stromal Cells Support LTC-ICs Better Than Bone Marrow-Derived Stromal Cells

Abstract 4804Quantification of putative hematopoietic stem cells (HSCs) in human bone marrow or cord blood is typically assessed by its potential to mediate long-term, multilineage engraftment when transplanted into immunodeficient murine recipients. However, in vitro surrogate assays are extremely attractive due to their relative ease of implementation, low cost and improved throughput of results. The long-term culture initiating cell (LTC-IC) assay serves this purpose by quantifying the ability of putative HSCs in a given population to be cultured for an extended period, typically 5 weeks. Previously, we have derived both mesenchymal stromal cells (MSCs) and endothelial cells (ECs) from human embryonic stem cells (hESCs). MSCs and ECs are important components of the osteogenic and vascular hematopoietic niches and hESC-derived stromal cells can provide autologous supporting cell populations for putative hESC-derived HSCs. Therefore, we evaluated if hESC-derived MSCs (hESC-MSCs) and hESC-derived ECs (hESC-ECs) would have improved ability to support LTC-IC activity of UCB and hESC-derived hematopoietic cells compared to MSCs isolated from human bone marrow (BM-MSCs) and a murine stromal line, M2-10B4. In the present study, we utilized LTC-IC culture of both hESC-derived CD34+CD45+ populations and UCB 34+ cells to test their LTC-IC potential on hESC-MSCs, hESC-ECs and a combination of the two hESC-derived populations. LTC-IC frequency and maintenance of input populations was assessed by survival/proliferation and preservation of colony forming cell (CFC) progenitors over 5 weeks. Initial studies showed all stromal layers were able to support short-term (3 week) culture of CFCs from CD34+ UCB. Total cell proliferation was found to be greatest with the hESC-derived stroma at 3 weeks. Additionally, hESC-MSCs and hESC-ECs yielded CFC comparable to M2-10B4 and significantly more than BM-MSCs. Co-culture of CD34+ UCB with a 1:1 mixture of hESC-MSCs and hESC-ECs yielded similar proliferation and CFC results to either stroma alone. After 5 weeks of culture to quantify LTC-ICs, both of the hESC-derived cells again supported expansion of UCB total cell numbers better than BM-MSCs and similar to M2-10B4. LTC-IC yield at 5 weeks was 85 ± 29 and 74 ± 12 per 103 input cells for hESC-MSC and hESC-EC, respectively, as compared to 15 ± 12 for BM-MSCs and 104 ± 56 for M2-10B4 co-culture. Combining the hESC-derived MSCs and ECs produced an LTC-IC yield of 54 ± 4. Next, we tested the ability of hESC-derived CD34+CD45+ cells to produce CFCs and LTC-ICs using co-culture with hESC-MSCs and M2-10B4. In short-term (2-3 week) assays, hESC-derived CD34+CD45+ cells declined in number to about 30% of the starting population on both stroma. However, between 3–5 weeks, cell number remained fairly constant on M2-10B4 while continuing to decrease on hESC-MSC. Interestingly, CFC yield for hESC-derived CD34+CD45+ cells at 2 weeks was 45 ± 43 per 103 input cells on M2-10B4, while co-culture with hESC-MSCs regularly failed to produce any CFC at this time. Between 3–5 weeks of culture no CFC or LTC-ICs were produced from hESC CD34+CD45+ using any of the stromal cells. Similar results were seen for hESC-derived CD34+CD43+ populations. Together, these results have two key findings. First, hESC-derived ECs and MSCs are effective in supporting LTC-ICs from UCB, and do so better than human BM-MSCs. Second, these same stromal cells do not support survival of hESC-derived LTC-ICs. In this manner, these results closely mimic studies that show limited long-term hematopoietic engraftment of hESC-derived cells when transplanted into immunodeficient mice. Therefore, this system can now be used to aid in identification of both hematopoietic niche elements and specific cell populations that mediate development and support of putative HSCs derived from hESCs and iPSCs. Disclosures:No relevant conflicts of interest to declare.

Distinct Functions of Stat5A and Stat5B in Chronic Myeloid Leukemia (CML): Stat5B Is Implicated in Survival and Self-Renewal and Stat5A in Imatinib Resistance.

AbstractAbstract 1214The use of tyrosine kinase inhibitors (TKI) has dramatically changed the prognosis and the natural history of CML. However the occurrence of drug resistances, especially at the stem cell level, requires the identification of novel functional targets. The signal transducer and activator of transcription 5 (Stat5), constitutively phosphorylated by BCR-ABL, is thought to be involved in the pathophysiology of CML, especially by its contribution to the antiapoptotic phenotype observed in CML cells via activation of Bcl-xL. There is currently no data linking clearly the involvement of Stat5 activation to TKI resistance in CML. Moreover, it has not been established, if the two closely related Stat5 factors, Stat5A and Stat5B, fulfill the same or distinct roles in leukemic cells. In order to study these potentially differential functions, we have specifically inhibited Stat5A and Stat5B through lentivirus-mediated RNA interference in human Ph+ CML cell lines K562, LAMA84 and Meg01 and their Imatinib mesylate (IM)-resistant counterparts. We have also transduced with the same lentiviral vectors, primary human CML CD34+ cells and evaluated the effects of Stat5A or B invalidation in terms of hematopoietic stem cell self-renewal in long-term culture initiating cell (LTC-IC) assays. In preliminary experiments, the successful inhibition of Stat5A or B was demonstrated using Western blotting with specific inhibition of each factor with the corresponding shRNA. In CML cell lines, Stat5B inhibition led to a massive apoptosis with growth arrest in all three cell lines whereas Stat5A inhibition had no effect. Surprisingly, Stat5B was active in the absence of canonical tyrosine phosphorylation and its absence strongly enhanced the apoptosis-inducing effects of TKI (Imatinib and Nilotinib). We then evaluated the role of Stat5A or B in the context of CML cell lines resistant to IM, in which there was no detectable ABL-kinase domain mutations neither MDR amplification. In this context, the Stat5A and B protein levels were identical but there was a strong over-activation of Stat5, as detected by Tyr694/699 phosphorylation status. In the two resistant sublines LAMA84-R and Meg01-R, anti-Stat5A and anti Stat5B selective immunoprecipitation assays further revealed that only Stat5A, and not Stat5B, was Tyr-phosphorylated. Therefore, in two independent IM-resistant sublines, LAMA84-R and Meg01-R, resistance correlated with over-activation of Stat5A, in the absence of canonical activation of Stat5B.We next investigated the role of Stat5A or B in primary CML stem cell self-renewal, using CD34+ cells from CML patients in chronic phase (CP-CML) at diagnosis. CD34+ cells were transduced with control (luciferase)-, Stat5A- or Stat5B-shRNA encoding lentiviral vectors and Long Term Colony Initiating Cell (LTC-IC) assays were performed. In four out of five patient samples, Stat5B-shRNA induced a massive inhibition of the LTC-IC activity. Stat5A-shRNA, on the reverse, had highly variable impact depending on the patients, either enhancing or somehow decreasing LTC-IC activity. We also performed similar assays with CD34+ progenitor cells collected from healthy donors. Except for one sample, Stat5B knock down had rather weak consequences on LTC-IC activity. Finally, to extend the potential roles of Stat5A and B in Imatinib resistance, we have transduced CD34+ cells of an IM-resistant CML patient whose cells had also been shown to be resistant to IM in vitro. GFP+ Stat5A-shRNA transduced cells were cell-sorted by FACS and their IM-resistance was tested in clonogenic assays. As compared to the control Luciferase shRNA vector, Stat5A inactivation reduced the colony numbers by 75%, with development of small sized colonies.Thus, these data strongly suggest that Stat5b plays a major role in the survival of CML cells, especially in the primitive hematopoietic cells, extending the results obtained in CML cell lines. Our data further show for the first time that this effect takes place in the absence of Tyrosine phosphorylation of Stat5B. Conversely, CML cells express tyrosine-phosphorylated Stat5A, whose levels strongly increased in TKI-resistant cells and its inhibition by an shRNA strategy leads to the reversal of TKI resistance of phenotype. Thus, Stat5A and Stat5B exhibit entirely distinct - not interchangeable- oncogenic activities in CML pathophysiology and represent novel therapeutic targets. Disclosures:No relevant conflicts of interest to declare.

Identification and Hematopoietic Potential of CD45 − Clonal Cells with Very Immature Phenotype (CD45 − CD34 − CD38 − Lin − ) in Patients with Myelodysplastic Syndromes

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38-Lin- cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34-CD38-Lin- cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site is a matter of debate, and CD45+CD34+CD38-Lin- HSCs were recently reported to be clonal. In the study reported here, we detected CD45-CD34-CD38-Lin- cells in the peripheral blood and bone marrow of patients with MDS and isolated them by successive application of density centrifugation, magnetic cell sorting, and fluorescence-activated cell sorting. Fluorescence in situ hybridization showed that CD45-CD34-CD38-Lin- cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-, they lacked CD117 and CD133 expression. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some patients. Freshly isolated CD45-CD34-CD38-Lin- cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When cocultured with stroma cells, CD45-CD34-CD38-Lin- cells showed only weak potential for proliferation and differentiation, yet they differentiated into CD34+ cells and then mature myeloid cells. This newly identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Identification and Hematopoietic Potential of CD45-Negative Clonal Cells with Very Immature Phenotype (CD45−CD34−CD38−Lin−) in Patients with Myelodysplastic Syndromes.

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Identification of a novel subpopulation of human cord blood CD34-CD133-CD7-CD45+lineage- cells capable of lymphoid/NK cell differentiation after in vitro exposure to IL-15.

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34(-) cell subsets that might reside at an earlier stage of differentiation than CD34(+) HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34(-)CD133(-)CD7(-)CD45(dim)lineage (lin)(-) HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133, 2) generation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates, 3) significant extended long-term culture-initiating cell activity, and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34(+)lin(-) cells, CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs maintained with IL-15, but not with IL-2 or IL-7, proliferated vigorously and differentiated into a homogeneous population of CD7(+)CD45(bright)CD25(+)CD44(+) lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRgamma genes, IL-15-primed CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs failed to achieve full maturation, as manifested in their CD3(-)TCRalphabeta(-)gammadelta(-) phenotype. Conversely, culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively, CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34(-)lin(-) HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined.

Stromal cells retard the differentiation of CD34+CD38low/neg human primitive progenitors exposed to cytokines independent of their mitotic history

AbstractStem cell proliferation induced by potent cytokines usually leads to a loss of primitive potential through differentiation. In this study, the ability of cytokines and murine MS5 stromal cells to independently regulate the proliferation and long-term culture-initiating cell (LTC-IC) activity of primitive CD34+CD38low/neg human bone marrow cells was evaluated. To compare populations with identical proliferation histories, cells were labeled with carboxy fluorescein diacetate succinimidyl ester, and LTC-IC activity was assessed 4 days later in cells that had accomplished the same number of divisions with or without MS5 cells. MS5 cells counteracted dramatically the loss of LTC-IC activity observed in the presence of cytokines alone. Thus, in the presence of MS5 cells, means of 1233 (n = 5) and 355 (n = 9) LTC-IC–derived colony-forming cells (CFCs) were generated by 1000 cells that performed 3 and 4 divisions respectively, whereas 311 (n = 5) and 64 (n = 5) CFCs were generated by 1000 cells cultured without MS5 cells. Interestingly, MS5 cells had no detectable effect on the LTC-IC activity of cells that divided only twice in 4 days—1606 CFCs (n = 6) and 1993 (n = 6) CFCs, respectively, without and with MS5 cells—and a 48 additional hours of coculture were necessary to unmask changes in the LTC-IC activity mediated by stromal cells. These results indicate that cytokines and stroma-derived signals can regulate independently the proliferation and differentiation of primitive cells and that these stroma-derived extracellular factors act directly on their target cells.

Expression of interferon-γ by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity

Several lines of evidence suggest that overexpression of interferon γ (IFN-γ) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-γ by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-γ. The effect of IFN-γ on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-γ vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-γ–expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-γ or control vector. Marrow cells transduced with either vector were transplanted into W/W v recipient mice. The percentage of vector-containing cells in the mIFN-γ mice was significantly lower than in the control mice, suggesting that mIFN-γ–transduced primitive cells may not have survived culture, or that mIFN-γ directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-γ–transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-γ group. These data indicate that ectopic or overexpression of mIFN-γ, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo. Published by Elsevier Science Inc.