Human CD34(lo)CD133(lo) fetal liver cells support the expansion of human CD34(hi)CD133(hi) hematopoietic stem cells.
ABSTRACT: We have recently discovered a unique CD34(lo)CD133(lo) cell population in the human fetal liver (FL) that gives rise to cells in the hepatic lineage. In this study, we further characterized the biological functions of FL CD34(lo)CD133(lo) cells. Our findings show that these CD34(lo)CD133(lo) cells express markers of both endodermal and mesodermal lineages and have the capability to differentiate into hepatocyte and mesenchymal lineage cells by ex vivo differentiation assays. Furthermore, we show that CD34(lo)CD133(lo) cells express growth factors that are important for human hematopoietic stem cell (HSC) expansion: stem cell factor (SCF), insulin-like growth factor 2 (IGF2), C-X-C motif chemokine 12 (CXCL12), and factors in the angiopoietin-like protein family. Co-culture of autologous FL HSCs and allogenic HSCs derived from cord blood with CD34(lo)CD133(lo) cells supports and expands both types of HSCs.These findings are not only essential for extending our understanding of the HSC niche during the development of embryonic and fetal hematopoiesis but will also potentially benefit adult stem cell transplantations in clinics because expanded HSCs demonstrate the same capacity as primary cells to reconstitute the human immune system and mediate long-term hematopoiesis in vivo. Together, CD34(lo)CD133(lo) cells not only serve as stem/progenitor cells for liver development but are also an essential component of the HSC niche in the human FL.
Project description:The identification of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34(-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34(+)CD38(-) SRCs. However, the purification level of rare CD34(-) SRCs in 18 lineage marker-negative (Lin(-)) CD34(-) cells (1/1000) is still very low compared with that of CD34(+)CD38(-) SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34(-) SRCs. Using 18Lin(-)CD34(-) cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34(-) SRCs and succeeded in highly purifying primitive human CD34(-) HSCs. The limiting dilution analysis demonstrated that the incidence of CD34(-) SRCs in 18Lin(-)CD34(-)CD133(+) cells was 1/142, which is the highest level of purification of these unique CD34(-) HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin(-)CD34(-) cells, as well as 18Lin(-)CD34(+) cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34(+) and CD34(-) SRCs.
Project description:The cell surface marker CD133 has been used to describe a revised model of adult human hematopoiesis, with hematopoietic stem cells and multipotent progenitors (HSCs/MPPs: CD133(+)CD45RA(-)CD34(+)) giving rise to lymphomyeloid-primed progenitors (LMPPs: CD133(+)CD45RA(+)CD34(+)) and erythromyeloid progenitors (EMPs: CD133(low)CD45RA(-)CD34(+)). Because adult and fetal hematopoietic stem and progenitor cells (HSPCs) differ in their gene expression profile, differentiation capabilities, and cell surface marker expression, we were interested in whether the reported segregation of lineage potentials in adult human hematopoiesis would also apply to human fetal liver. CD133 expression was easily detected in human fetal liver cells, and the defined hematopoietic subpopulations were similar to those found for adult HSPCs. Fetal HSPCs were enriched for EMPs and HSCs/MPPs, which were primed toward erythromyeloid differentiation. However, the frequency of multipotent CD133(+)CD45RA(-)CD34(+) HSPCs was much lower than previously reported and comparable to that of umbilical cord blood. We noted that engraftment in NSG (NOD scid gamma [NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ]) mice was driven mostly by LMPPs, confirming recent findings that repopulation in mice is not a unique feature of multipotent HSCs/MPPs. Thus, our data challenge the general assumption that human fetal liver contains a greater percentage of multipotent HSCs/MPPs than any adult HSC source, and the mouse model may have to be re-evaluated with respect to the type of readout it provides.
Project description:Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.
Project description:A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133(+)CD34(+) cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133(+)CD34(+) progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133(low)CD34(+) cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.
Project description:The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133(hi)/ER(lo) cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133(hi)/ER(lo)/IL6(hi) cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133(hi)/ER(lo)/OXPHOS(lo). These cells exit metabolic dormancy via an IL6-driven feed-forward ER(lo)-IL6(hi)-Notch(hi) loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133(hi) CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133(hi)/ER(lo) cells mediating metastatic progression, which is sensitive to dual targeted therapy.
Project description:We previously identified CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells as primitive hematopoietic stem cells (HSCs) in human cord blood. In this study, we develop a prospective ultra-high-resolution purification method by applying two positive markers, CD133 and GPI-80. Using this method, we succeed in purifying single long-term repopulating CD34- HSCs with self-renewing capability residing at the apex of the human HSC hierarchy from cord blood, as evidenced by a single-cell-initiated serial transplantation analysis. The gene expression profiles of individual CD34+ and CD34- HSCs and a global gene expression analysis demonstrate the unique molecular signature of CD34- HSCs. We find that the purified CD34- HSCs show a potent megakaryocyte/erythrocyte differentiation potential in vitro and in vivo. Megakaryocyte/erythrocyte progenitors may thus be generated directly via a bypass route from the CD34- HSCs. Based on these data, we propose a revised road map for the commitment of human CD34- HSCs in cord blood.
Project description:Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.
Project description:The CD133(hi) sub-population of prostate epithelial cells has been demonstrated to possess tumor-initiating capacity consistent with that of the cancer stem cell theory. However, the involvement of oncogenes such as c-myc has not been fully elucidated in the CD133(hi) sub-population.We have isolated primary prostate cell strains (IBC-10a) and immortalized them by transfection with hTERT. The in vitro and in vivo tumorigenic capacity of isolated CD133(hi) and CD133(lo) cells was evaluated with respect to c-myc expression using specific sense and anti-sense oligonucleotides.Freshly immortalized cells consisted of <3.3% CD133(hi)/CD24(hi) sub-population (SP). "Prostaspheres" generated from single CD133(hi) cells in the presence of EGF consisted of approximately 10% CD133(hi) SPs in 12-21 day cultures. A single Prostasphere generated from single CD133(hi) cells (6-10 cell stage at day 6 injected i.t.) produced dysplastic lesions in NOD-SCID mice (n = 4/5). Treatment of Prostaspheres from CD133(hi) SPs in vitro with c-myc or cyclin D1 anti-sense oligonucleotides totally blocked colony forming ability and growth. Furthermore, treatment of fully formed, 6-day Prostaspheres for 48 hr with c-myc anti-sense significantly reduced c-myc expression and their ability to generate lesions in NOD-SCIDs (n = 10 Prostaspheres injected i.t./mouse).These data demonstrate for the first time that a single CD133(hi) cell is competent to generate Prostaspheres in vitro and that CD133(hi) Prostaspheres require c-myc to grow and form dysplastic lesions in vivo.
Project description:MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.