ALBERT

Description: Abstract 576 Hematopoietic stem cells (HSCs) represent a rare subset of bone marrow (BM) cells in adult mice that are ultimately responsible for the replenishment of all different mature blood cell types through a hierarchically organized cascade of differentiation steps. Although HSCs were previously considered a functionally and phenotypically homogeneous population, recent studies tracking the 4 to 6 month progeny of a large number of highly purified, individually transplanted, single mouse HSCs and their serially generated derivatives in sublethally irradiated hosts clearly reveal a functional heterogeneity of HSCs. The results have revealed 2 distinct and stably propagated clonal subtypes of HSCs with unrestricted self renewal activity that we have termed α- and β-HSCs. β-HSCs are those that show a relatively balanced output of mature myeloid and lymphoid cells. α-HSCs have an equivalent self-renewal and myelopoietic activity to β-HSCs but, in contrast, are characterized by a variable and often extreme failure to produce mature lymphoid cells. Since it is well established that the reduced activity of the immune system with aging corresponds with a decrease in the frequency and activity of both B- and T lymphocytes and their respective progenitors, it is of interest to determine whether this simply represents a generalized aging of the hematopoietic system or rather an age-related change in the composition of the HSC compartment. The most rigorous approach to determine the time of appearance and kinetic changes in the distribution of different types of HSCs during development and aging is to study single-cell transplants and their clonal progeny using an ontogeny-independent HSC purification scheme. We recently showed that the E-SLAM (CD45+ Endothelial protein receptor EPCR+ CD150+ CD48−) phenotype could be used to achieve high HSC purity across development (E14.5 fetal liver, 3-week BM, 4-week BM, 10–12 week BM and aged BM) despite known ontological differences in HSC proliferative activity between these populations. These highly-purified populations of HSCs can thus be utilized to prospectively isolate and singly transplant these HSCs to enable study of their clonally progeny. Clonal analysis of the E-SLAM HSC compartment of aged mice showed an increase in frequency of α-HSCs. To investigate the mechanism underlying the lymphopoietic deficiency characteristic of α-HSCs, we undertook a set of experiments to compare the in vivo generated progeny of α- vs β-HSCs. We particularly focused on a quantitative and qualitative analysis of the common lymphoid progenitor (CLP) compartment (Lin− CD127+ CD117low Sca1low). In contrast to previous reports of a decrease in CLP numbers in old mice, we simply found a broader distribution of CLPs in old mice. We did however find a decreased number of CLPs generated per HSC measured as the CLP/LSK ratio (Lin− CD127− CD117+ Sca1+) with age suggesting that – if the aged BM is dominated by α-HSCs – that α-HSCs possess a quantitative defect in CLP production. Intriguingly, the number of CLPs derived from mice reconstituted with an α stem cell were significantly reduced in comparison to CLPs derived from β-HSCs. To measure the B-cell production activity of CLPs produced from α- vs β-HSCs we utilized the OP9 co-culture system and plated limiting numbers of CLPs derived from reconstituted mice which were then compared to the CLP productivity of young and old CLPs from non-reconstitued mice. Functional analysis of CLPs from individual young mice showed a consistent B-cell frequency of 1/3.6 (n=13) in the OP9 co-culture system whereas CLPs from old mice showed either good B-cell potential comparable to young CLPs or very reduced B–cell potential. CLPs derived from β-HSCs show a consistent B-cell potential (1/11.3, n=9) even after reaching a physiological age of 〉100wks whereas when CLPs could be detected from α-HSCs they showed a reduced B-cell activity in vitro. The results of our study demonstrate that the decrease in lymphoid activity during aging is likely caused by the relative increase in α-HSCs which have both a quantitative and qualitative CLP defect. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150+ subset of the EPCR+CD48−CD45+ fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150− subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.

Description: Abstract 45 Fetal hematopoietic stem cells (HSCs) in mice differ from their adult counterparts in a number of key properties. These include a higher cycling activity, an ability to more rapidly reconstitute the HSC compartment of irradiated recipient mice, a higher output of myeloid as compared to lymphoid progeny, and a greater sensitivity to the self-renewal promoting activity of Steel factor. We have previously shown that most of these features of fetal HSCs are sustained until 3 weeks after birth at which time they are rapidly (within 1 week), completely and permanently replaced with the corresponding properties of adult HSCs. A candidate regulator of this transition, Hmga2, was identified based on its greater expression in highly purified fetal versus adult HSCs (CD45+EPCR+CD48−CD150+; E-SLAM cells) with persistence of this difference in the matching lineage-negative (lin−) compartments. Experiments in which Hmga2 was overexpressed by lentiviral transduction of purified adult HSCs which were then transplanted into irradiated mice provided evidence that this chromatin remodeling factor can activate a fetal-like HSC program in these cells; i.e., more rapidly reconstitute the HSC compartment (increased self-renewal response) and produce clones with a higher proportion of myeloid cells. Based on the known ability of the let-7 family of microRNAs (miRNAs) to target Hmga2 transcripts resulting in their degradation and/or translational repression, we next hypothesized that let-7 miRNAs might be involved in controlling HSC developmental programs. A comparison of the levels of expression of 6 members of the let-7 family in purified fetal and adult HSCs, as well as in lin− hematopoietic cells, showed that transcripts for all of these are higher in the adult subsets, although this difference was significant only for let-7b (p

Description: Significant advances have been made in the development of methods for purifying murine hematopoietic cells with longterm (〉4 months) in vivo reconstituting ability although these longterm repopulating cells (LTRCs) remain heterogeneous with regard to the self-renewal (SR) activity they display when transplanted into irradiated hosts. Furthermore our group has also identified cell culture conditions that differentially alter LTRC activity without immediate effects on their proliferation or survival. Here, we show that highly purified LTRCs with high and low SR properties can be prospectively isolated from normal adult mouse bone marrow (ABM) as 2 separate populations according to their expression of CD150 within the EPCR++CD48−CD45mid fraction of cells: 56% total LTRCs and 43% of the high SR type in the CD150+ subset vs. 39% total LTRCs and 32% of the low SR type in the CD150− subset (as determined from 62 and 28 single cell transplants, respectively). As a first test of whether these populations would likely be useful to search for new molecular differences associated with their different SR properties, we compared the level of expression in these 2 populations of a small set of genes previously reported to regulate LTRC SR activity: c-Kit, Bmi1, Gata3, Rae28, Ezh2 and Lnk by quantitative real-time PCR (Q-RT-PCR). This exercise revealed transcript levels of the first 4 of these genes to be significantly higher in the CD150+ subset that is selectively enriched in high SR LTRCs, thus validating the concept that they have a distinct molecular signature. Previous evidence shows that high SR LTRCs are present in both FL LTRCs and ABM LTRCs but they differ in some properties (i.e.: cell cycle status, regeneration kinetics). We therefore began a search for ontogeny-independent components of the SR machinery by comparing tags present in 2 LongSAGE libraries produced from CD45midlin−Rho−SP ABM cells and from lin−Sca1+CD43+Mac1+ embryonic day 14.5 fetal liver (FL) cells (each 20–30% total LTRCs and 12–20% of the high SR type, as determined by 132 (FL) and 352 (ABM) single cell transplants, respectively). From these comparisons and additional data in other publicly available datasets for primitive murine hematopoietic cells, we identified 28 genes not previously shown to have a functional role in LTRC SR control. We then compared the level of expression of these 28 genes between the CD150+ subsets of EPCR++CD48−CD45mid ABM cells and FL cells (24% total LTRCs and 12% high SR LTRCs in the FL subset) and their respective downstream lin− progeny. This comparison revealed 10 of these genes to be down-regulated in the lin− populations of both ABM and FL. Further comparison of the expression of these 10 genes between the high vs. low SR LTRCs (found in the CD150+ and CD150− subsets of EPCR++CD48−CD45mid) ABM cells showed the expression of 5 (Vwf, Rhob, Pld3, Prnp and Smarcc2) to be downregulated in the CD150− (low SR LTRC) subset. Interestingly, the first 4 of these genes, as well as 2 of the preliminary set of SR regulators (Bmi1 and Gata3), were also selectively down-regulated in EPCR++CD150+CD48−CD45mid ABM cells that had been incubated for 16 hours in 1 or 10 ng/ml Steel factor + 20 ng/ml IL-11 (conditions that decrease LTRC activity in vivo 4–5-fold before any of these divide or die). Taken together, these results point to the existence of more, although a rather small number of additional genes, including Vwf, Rhob, Pld3, and Prnp, whose products may be involved in controlling the SR potential of normal mouse LTRCs.

Description: Abstract 2634 Fetal and early neonatal hematopoietic stem cells (HSCs) are distinct from their adult counterparts by their rapid turnover and expansion rates in vivo. However, the mechanisms underlying the regulation of these properties are poorly understood. In previous studies using serial limiting-dilution competitive repopulating transplant assays, our lab has shown that the rapid expansion phenotype of fetal HSCs is at least partially intrinsically determined since significantly more daughter HSCs are produced from fetal as compared to adult HSCs when similar numbers are transplanted into the same type of irradiated adult host. Additionally, we have observed a conversion of fetal HSCs to the adult regeneration phenotype that occurs within six weeks of transplantation in the primary host. To facilitate a comparison of highly-purified subsets of fetal and adult HSCs identified by an identical phenotype, we adopted the use of the CD45+EPCR+CD150+CD48− (E-SLAM) phenotype which we found gave HSC purities of 20–50% for hematopoietic tissues from early fetal to aged adulthood. We then used comparative gene expression analysis to identify candidate regulators of the fetal HSC high self-renewal program. This gave 20 candidate genes whose transcript levels were measured by quantitative real time PCR in E-SLAM cells isolated from E14.5 fetal liver (FL) and adult bone marrow (ABM). Of these genes only Hmga2 and Smarcc1 showed significant differences (p

Description: MicroRNAs (miRNAs) have been shown to be developmental regulators in various organisms and tissues such as the hematopoietic system. miRNA profiling studies have been primarily performed on specific aspects of hematopoiesis like lymphocyte or red blood cell development. However, a comprehensive study including rare hematopoietic stem cell populations and various lineages has yet to be published. MiRNA expression profiling within the hematopoietic tree is challenging due to difficulties in obtaining highly purified samples of stem and progenitor cell populations as well as the high cost and labour associated with global profiling approaches. The combined requirements of high sensitivity, dynamic range and efficient throughput pose serious obstacles to the use of established methods including Northern Blot, cloning, miRNA microarrays, and deep sequencing. Real time PCR offers the requisite dynamic range and sensitivity, but is labour intensive and prohibitive in cost using conventional formats. To overcome these limitations we combined new high throughput microfluidic technologies with a 288-plex real time PCR approach to quantify miRNAs in hematopoietic stem cells and lineage positive cells. This approach allowed us the simultaneous detection of 288 miRNAs in small numbers (≤3000) of cells across multiple subpopulations of the murine hematopoietic tree. Twenty unique murine hematopoietic cell populations were isolated through current FACS sorting strategies, including hematopoietic stem cells (HSCs) based on SLAM and LSK markers, myeloid and lymphoid progenitor cells as well as mature populations of all lineages. The cells were immediately lysed after sorting and reverse transcribed using 3 pools of 96 stemloop RT-primers, followed by a PCR pre-amplification step. In order to detect individual amplified products, we used BioMark™ 48.48 Dynamic Arrays (Fluidigm Corp, San Francisco, USA) and miRNA specific TaqMan probes. For each miRNA, a series of synthetic miRNA dilutions was used as a standard to determine the absolute number of miRNA molecules per cell. This analysis further revealed systematic and miRNA-specific variations in the sensitivities of Taqman assays, highlighting that RT-PCR analysis without the inclusion of an absolute standard may be misrepresentative of the true molecular abundance. Hierarchical clustering analysis and comparison between hematopoietic stem cell (HSC) populations and mature populations revealed miRNAs that are critical for hematopoietic development and maturation. In general, miRNAs detected at the highest abundance were miR-706 and miR-720, which is likely due to highly reactive Taqman assays for these targets. Of the tested 288 miRNAs, only 133 were detected across all cell populations. Most of these miRNAs exhibited a mixed expression profile, with expression peaks in the differentiated populations. Consistent with previous results, we detected a strong increase of miR-223 within myeloid differentiated populations. The highest levels were detected in neutrophils and monocytes, but surprisingly low levels were found in mast cells, supporting the specific role of miR-223 in myeloid differentiation. Other miRNAs highly enriched in differentiated cells were miR-142 and miR-16. Clustering revealed a distinct miRNA expression pattern for the profiled HSC populations including miRNAs located in the Hox cluster and the miR-181 family. In conclusion, we applied a novel technical approach to quantify a broad range of miRNAs in rare cell populations. With this approach we can further define the expression patterns of miRNAs from hematopoietic stem cells to mature lineages.

Description: Abstract 1271 Recent tracking of the primitive and mature progeny of more than 500 single cell transplants of highly purified hematopoietic stem cells (HSCs) in cell suspensions isolated from the bone marrow of young adult mice (8–12 weeks old) has shown that this pool is intrinsically heterogeneous. This heterogeneity is manifested as differences both in the durability (beyond 6 months) of the self-renewal activity of the HSCs detected in irradiated recipients as well as in the specific lineage contributions maintained in secondary and tertiary recipients of cells generated within each clone. Interestingly, durable HSC self-renewal activity (serial transplantability) was found to be exclusively associated with a robust and sustained ability to produce myeloid cells, regardless of their ability to produce lymphoid progeny at any stage of lymphoid progenitor development. Thus 2 subtypes of HSCs with durable self-renewal activity could be distinguished depending on whether the sustained myelopoietic activity was accompanied by an equivalent robust lymphoid differentiation activity (β-HSCs), or not (α-HSCs). Preliminary examination of fetal liver HSCs with durable self-renewal activity showed that this compartment is dominated by β-HSCs in contrast to the marrow of young adult mice where ∼30% of this HSC pool are α-HSCs. Since previous studies have documented a switch between 3 and 4 weeks after birth in several key properties of HSCs (indicative of an abrupt change from a fetal to an adult HSC program), we were interested in the question of whether the appearance of α-HSCs might be another aspect of this switch or, alternatively, whether the onset of hematopoiesis in locations other than the fetal liver (e.g., the marrow and spleen) might be a contributing factor. Accordingly, we initiated experiments to examine the proportional representation and absolute number of cells with an E-SLAM phenotype (CD45+EPCR+CD48−CD150+) and their functional activity (4-month repopulation of irradiated mice) specific for HSCs in tissues where hematopoiesis is seen during different stages of development between E14.5 and 4 weeks after birth. The number of E-SLAM cells increased ∼15 fold in the fetal liver between E14.5 and E18.5 by which time they were also present in the marrow and spleen but altogether comprising only ∼5% of the fetal liver compartment (assuming the femurs, tibiae and pelvis represent ∼30% of the total marrow). Analysis of the clonal outputs obtained in vivo from single E-SLAM cell transplants from each of these sources showed that α-HSCs are already present as a rare subet of all HSCs in the E14.5 liver and amplify equally with β-HSCs maintaining a ratio of 1:10 α-HSCs to β-HSCs in this organ. Interestingly, despite the small numbers of E-SLAM cells in the E18.5 marrow, these contained readily detectable HSCs in transplant assays and showed a ratio of α-HSCs to β-HSCs of 1:2. This ratio was mirrored in the marrow of adult mice and was also similar to that seen in the marrow and spleen of 3 and 4 week-old mice. These findings show that α-HSCs, by comparison to β-HSCs, have a greater ability to localize in the marrow which appears to then selectively promote the expansion of α-HSCs. These results also show that the initial appearance and early selective expansion of α-HSCs during fetal and early post-natal life is unlikely to be regulated by the same mechanisms that impose an adult program on murine HSCs between 3 and 4 weeks after birth. Rather these results favor a separate, intrinsically determined developmentally controlled mechanism that endows α-HSCs with a higher self-renewal probability, a more rapid turnover, a lower apoptotic activity, or a combination of these differences as compared to β-HSCs. This would explain the initial rapid and marked selective expansion of α-HSCs that occurs during the late fetal and early neonatal period of high HSC proliferation which extends to 3 weeks after birth and is then followed by a much slower rate of selective expansion of α-HSCs during adulthood when the turnover of HSCs is minimal. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (〉20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (〉1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. Disclosures: No relevant conflicts of interest to declare.