Human Human Hematopoietic Stem Cells Research Articles

CD34− Cells at the Apex of the Human Hematopoietic Stem Cell Hierarchy Have Distinctive Cellular and Molecular Signatures

In addition to well-characterized CD34(+) hematopoietic stem and progenitor cells (HSPCs), the human hematopoietic stem cell (HSC) hierarchy contains a rare CD34(-) population with severe combined immunodeficiency-repopulating capacity. However, little is known about the molecular characteristics of these CD34(-) cells or their relationship to the CD34(+) populations. Here, we show that the self-renewing Lin(-)CD34(-)CD38(-)CD93(hi) population contains cells that not only function as HSCs, but can also be placed above the CD34(+) populations in the hematopoietic hierarchy. These cells have an active Notch pathway, in which signaling through Delta4 is crucial for maintenance of the primitive state, and combined signals from Jagged1 and TGF-β are important in controlling its quiescence. They are also refractory to proliferative signals and show a repressed canonical Wnt pathway, in part regulated by Notch. Overall, therefore, CD34(-) cells represent an immature and quiescent human HSC population maintained through a distinctive network of cellular signaling interactions.

542. Transduction of Murine, Nonhuman Primate, and Human Hematopoietic Stem Cells (HSCs) by Recombinant AAV Serotype Vectors: Identification of AAV6 as the Most Efficient Serotype for Human HSCs, and Further Augmentation in Transduction Efficiency In Vitro and In Vivo with Point-Mutations of Tyrosine Residues in the Viral Capsid

542. Transduction of Murine, Nonhuman Primate, and Human Hematopoietic Stem Cells (HSCs) by Recombinant AAV Serotype Vectors: Identification of AAV6 as the Most Efficient Serotype for Human HSCs, and Further Augmentation in Transduction Efficiency In Vitro and In Vivo with Point-Mutations of Tyrosine Residues in the Viral Capsid

CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells

Pentatransmembrane glycoprotein prominin-1 (CD133) is expressed at the cell surface of multiple somatic stem cells, and it is widely used as a cell surface marker for the isolation and characterization of human hematopoietic stem cells (HSCs) and cancer stem cells. CD133 has been linked on a cell biological basis to stem cell-fate decisions in human HSCs and emerges as an important physiological regulator of stem cell maintenance and expansion. Its expression and physiological relevance in the murine hematopoietic system is nevertheless elusive. We show here that CD133 is expressed by bone marrow-resident murine HSCs and myeloid precursor cells with the developmental propensity to give rise to granulocytes and monocytes. However, CD133 is dispensable for the pool size and function of HSCs during steady-state hematopoiesis and after transplantation, demonstrating a substantial species difference between mouse and man. Blood cell numbers in the periphery are normal; however, CD133 appears to be a modifier for the development of growth-factor responsive myeloerythroid precursor cells in the bone marrow under steady state and mature red blood cells after hematopoietic stress. Taken together, these studies show that CD133 is not a critical regulator of hematopoietic stem cell function in mouse but that it modifies frequencies of growth-factor responsive hematopoietic progenitor cells during steady state and after myelotoxic stress in vivo.

CD166 (ALCAM): A Functional Marker of Primitive Murine and Human Hematopoietic Stem Cells and Cellular Elements of Their Niche

AbstractAbstract 640Phenotypic definition of murine and human hematopoietic stem cells (HSC) relies on a large number of markers. Few surface antigens with functional importance have been identified as key common markers on adult murine and human HSC. Intimate interactions between HSC and elements of the hematopoietic niche (HN) depend on ligand-counter ligand molecules co-expressed independently on niche components or putative HSC. To date, not a single surface marker has been identified as a common marker expressed on murine and human HSC and on cells of the HN. We previously demonstrated that CD166 is expressed on osteoblasts (OB) that mediate a hematopoiesis enhancing activity (HEA). Given that CD166 is a member of the immunoglobulin superfamily that can mediate homophilic cell-cell interactions, we investigated the role of CD166 in identifying HSC and the impact of CD166 on hematopoiesis, stem cell engraftment, and the HN. Interestingly, CD166+, but not CD166- fractions of murine and human repopulating HSC identified by a rigorous hierarchical classification for each species mediated robust long-term engraftment. In the murine system, 25 sorted Lineage- Sca1+ ckit+ (LSK) CD48-CD150+CD166+ cells mediated 69.5 ± 7.3% chimerism 4 months post-transplantation (PT) while donor-derived chimerism supported by 25 sorted LSKCD48-CD150+CD166- cells was only 13.6 ± 11.6% (p<0.01) suggesting that CD166 identifies long-term repopulating cells beyond what is possible with SLAM markers. In the human system, 1000 cord blood-derived Lin-CD34+CD38-CD49f-CD166+ cells and Lin-CD34+CD38-CD49f+CD166+ cells engrafted at 44.5 ± 9.7% and 38.4 ± 8.9%, respectively 16 weeks PT in conditioned NSG mice. More importantly, chimerism derived from Lin-CD34+CD38-CD49f+CD166- cells was 1.6 ± 0.1% (p<0.01 vs both fractions) demonstrating that the CD166+ but not the CD166- fraction of CD34+Lin-CD38- cells (regardless of the status of CD49f expression) contains long-term engrafting human HSC. In CD166 knockout (KO) mice, numbers of LSKCD48-CD150+ cells in the bone marrow and Lin-CD48- cells in the peripheral blood were significantly reduced relative to wild-type (WT) controls although other hematopoietic parameters in KO mice were within normal ranges. Phenotypically defined HSC from CD166−/− mice failed to engraft in lethally irradiated WT recipients. Levels of engraftment 4mo PT of 1,000 LSK cells from WT donors into WT recipients was 71.8% ± 8.3% while that obtained from a similar number of KO cells was 5.8% ± 2.8% (p<0.01). To permit direct comparison of KO and WT mice as recipients, both genotypes were transplanted with purified LSK cells from GFP C57BL/6 mice. While short-term repopulating GFP cells engrafted efficiently in KO mice 1mo PT (66.0% ± 6.5%), reconstitution declined substantially 2mo PT and was 10.3% ± 2.7% at 3mo PT (compared to 52.6% ± 10.4% in WT hosts, p<0.01) and less than 5% at 4mo PT, demonstrating that the CD166−/− hematopoietic niche can not support long-term repopulating cells. We used our previously described co-culture system to assess the impact of homophilic CD166 interactions on the HEA of OB. The highest HEA was reached when both OB and LSK cells expressed CD166. However, when either or both cell types lacked CD166 expression, the degree of HEA was significantly lower demonstrating that homophilic CD166 interactions are critical to maintaining HSC function. Since Stat3 has 3 binding sites on the CD166 promoter, we examined the relationship between expression of Stat3 and CD166. HSC from Stat3−/− mice which do not engraft efficiently in WT recipients expressed very low levels of CD166. In addition, pharmacologic inhibition of Stat3 expression led to a simultaneous inhibition of CD166 expression. Reconstitution kinetics data and survival of KO mice under hematopoietic stress conditions suggested that CD166−/− HSC have an intrinsic self-renewal capacity precluding them from both rapid proliferation and expansion and maintenance of the stem cell pool in the HN. Our data illustrate for the first time, that CD166 is a universal marker of both murine and human HSC and OB within the HN and suggest that CD166 may modulate HSC-niche interactions and impact stem cell fate. The conserved homology between murine and human CD166 provides an excellent bridge between human and murine studies for efficient translational investigations and interventions for enhancing stem cell engraftment and clinical utility. Disclosures:Broxmeyer:CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Improved Engraftment of Human Hematopoietic Stem Cells in NOD/SCID Mice by the Antioxidant, N-Acetyl-L-Cysteine

Abstract 1884Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice are widely used as a human-mouse xeno-transplant model for assessing engraftment of human hematopoietic stem cells (HSCs). Optimizing this model and understanding the variables that affect engraftment are critical to correctly interpret the engraftment results of human HSCs in NOD/SCID recipients. However, the engraftment efficiency of human HSCs remains very low in NOD/SCID mice. Larger efforts have been devoted to improving engraftment by increasing the immune-deficiency of the mice, but less effort has been made toward other negative parameters in the host. Our preliminary study showed that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in the bone marrow (BM) in comparison with other commonly used mouse strains (C57BL/6 and BALB/C). Given the previous studies by us and others showing that excessive ROS could impair the function of HSCs and that antioxidants were able to overcome the exhaustion of mouse HSCs in transplant recipients, we hypothesized that the poor engraftment of human hematopoietic cells in NOD/SCID recipients may be partially attributed to higher levels of ROS in NOD/SCID BM and a reduction of ROS by antioxidants may improve the engraftment of human HSCs in NOD/SCID mice.To test this hypothesis, NOD/SCID (8- to 12-week-old) mice were injected subcutaneously daily with an antioxidant, N-acetyl-L-cysteine (NAC) or PBS (control) for two weeks before being irradiated with 200 cGy from a cesium-137 source at 70cGy/min. Different doses of CD34+ cells or highly-enriched HSCs from human cord blood (CB) were injected through the tail vein or into the right tibia of the mice. The mice were maintained on NAC treated drinking water following injection, then sacrificed 12–14 weeks after transplantation to measure the engraftment levels of different hematopoietic cell lineages.We found that treatment with NAC was able to lower the levels of ROS in NOD/SCID BM. At the highest dose of injected CD34+ cells (>5×105), the NAC treated recipients displayed a significant increase of engraftment (2.1-fold) when compared with the control group (Control vs. NAC treated recipients: 11.04±3.11% vs. 23.21±4.0%, p=0.0224; n=20/each). This improvement was even more significant when injected cell numbers were reduced (2×105 and 1×105 had 3.9- and 4.9- fold higher engraftment, respectively, in NAC treated recipients), thus suggesting that saturating levels of HSCs may ease the anti-oxidant effect on engraftment. Furthermore, we also demonstrated higher levels of overall engraftment and multi-lineage differentiation of human HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+Rholow) with a limiting dilution analysis. In comparison with the control mice, NAC treated recipients displayed 2.5-, 3.5-, and 5.7-fold increases in engraftment in the injected tibia (IT), BM and spleen, respectively. The frequency of SCID-repopulating cell (SRC) in IT was approximately 3.0-fold higher in NAC treated mice than in control mice (1 in 108 vs. 1 in 36). Similar improvements (4.1- to 7.9- fold) in SRC frequencies were also detected in BM and spleen. Notably, NAC increased the probability of positive engraftment when a single human HSC was directly transplanted into the BM of NOD/SCID mice. In summary, our current study uncovers a previously unappreciated negative effect of ROS in the human-NOD/SCID xenotransplant model and reduction of ROS via antioxidants such as NAC may significantly enhance the engraftment of human hematopoietic stem cells in NOD/SCID mice. Disclosures:No relevant conflicts of interest to declare.

Short Term Signalling Responses of the Most Primitive Subsets of Human Hematopoietic Cells Stimulated in Vitro Correlate with Their Subsequent Self-Renewal Behaviour.

AbstractAbstract 2341 Background:Growth factors (GFs) that stimulate the proliferation of human hematopoietic stem cells (HSCs) in vitro have been identified, but maintenance of the original stem cell potential has remained a challenge. We have confirmed the observation that UG26–1B6 cells produce factors that enhance the maximum yield in vitro of HSCs achievable with SCF, FLT3-L, IL-3, IL-6, and G-CSF (5 GFs) alone (∼5-fold increase in lympho-myeloid repopulating cells by limiting dilution transplant analysis in NOD/SCID-IL-2Rγc null mice assessed 20 weeks post-transplant). It was therefore of interest to determine whether these different outcomes could be correlated with specific signalling events. Methods:CD34+cell-enriched human cord blood (CB) cells were stimulated with 5 GFs ± UG26 conditioned medium (CM) for defined periods, and then fixed in paraformaldehyde, stained for surface markers, alcohol permeabilized and stained with antibodies specific for multiple intracellular signalling intermediates. Samples were then analyzed using either a CyTOF mass cytometer (34 unique parameters) or a LSR Fortessa cell analyzer (11 parameters). Data analysis was performed using a combination of Cytobank, SPADE, and R using the ’flowCore’, ’flowType’, ’lattice’, ’SamSpectral’, and ’gplots’ packages. Results:Analysis by CyTOF mass cytometry of 106 CD34+ CB cells after 15 minutes of their stimulation ± 5 GFs ± CM showed that the HSC-enriched subset (Lin−CD34+CD38−CD90+CD49f+cells) exhibited similar responses to the 5 GFs and 5 GFs+CM cocktails (average shifts in median intensity [Δasinh (stimulated-unstimulated)] of 0.32 [-0.14 to 1.96] and 0.37 [-0.02 to 1.78]) with increased phosphorylation of S6, STAT3, STAT5, CREB, IκBa (total protein), AKT, Syk/ZAP70, and ERK1/2. However, the addition of CM did selectively reduce the level of pS6 and pAKT seen with 5 GFs alone, and also caused an increase in pCREB. In addition, activation of pSyk/ZAP70 was detected only in HSCs stimulated with 5 GFs + CM. Phosphoflow analysis of the same subset of cells from a second CB pool confirmed the CyTOF results for pAKT, pSTAT5, pCREB and pERK1/2 (variably) following a 15 minute stimulation but not pSyk/ZAP70. After 2 hours of stimulation, the activation patterns were sustained for most targets for cells exposed to 5 GFs + CM but were reduced in 5 GFs alone - particularly pCREB which was decreased to levels in unstimulated cells. After 24 hours, pAKT and pSTAT5 showed continued activation in response to 5 GFs ± CM, whereas pCREB and pERK1/2 signal were reduced to below starting levels.Hartigans’ Dip Test for Unimodality revealed significant deviations from unimodality for the mass cytometry HSC data. Principal component analysis (PCA) of the HSC data followed by subsequent clustering also revealed a number of differentially responsive sub-populations including a broadly cytokine non-responsive, non-apoptotic (cleaved PARP−) population, as well as a minority population with high levels of pSrc and pPLCγ, but reduced activation of other pathways. Further analysis using SPADE revealed decreased cytokine responsiveness with higher CD49f intensity. Weak but significant negative correlations between CD49f and pSTAT3, pCREB, and pS6 and positive correlations with pSrc and pPLCγ (holm-adjusted p values <0.05; r values −0.12 – 0.38) were also observed.In the CyTOF experiment, multipotent progenitors (MPP; Lin−CD34+CD38−CD90−CD49f– cells) showed broadly similar patterns and levels of activation as HSCs when CM was added (p=0.44), but the median activation was attenuated in 5 GFs alone (p=0.048). MPPs also showed lower levels of multiple phosphorylation events, despite both HSC and MPP populations being Ki67−. This was consistent regardless of cell stimulation status, time point or measurement platform. Conclusions:We have discovered heterogeneity in the signal responses of highly purified fractions of very primitive subsets of human CB cells to culture conditions that affect their self-renewal differentially, that distinguish HSCs from MPPs, and that further subdivide the HSC subset. Among the events surveyed, differences in the timing and intensity of signal activation down common pathways were identified rather than any evidence of selective pathway utilization. These findings hold promise for the identification of how external factors modulate the fates of normal and transformed human HSCs when they are stimulated to divide. Disclosures:No relevant conflicts of interest to declare.

Implication of microRNAs in the Pathogenesis of MDS

MicroRNAs (miRNAs) are significant regulators of human hematopoietic stem cells (HSC), and their deregulation contributes to hematological malignancies. Myelodysplastic syndromes (MDS) represent a spectrum of hematological disorders characterized by dysfunctional HSC, ineffective blood cell production, progressive marrow failure, and an increased risk of developing acute myeloid leukemia (AML). Although miRNAs have been primarily studied in AML, only recently have similar studies been performed on MDS. In this review, we describe the normal function and expression of miRNAs in human HSC, and describe mounting evidence that deregulation of miRNAs contributes to the pathogenesis of MDS.

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

Rapid generation of human B-cell lymphomas via combined expression of Myc and Bcl2 and their use as a preclinical model for biological therapies

Although numerous mouse models of B-cell malignancy have been developed via the enforced expression of defined oncogenic lesions, the feasibility of generating lineage-defined human B-cell malignancies using mice reconstituted with modified human hematopoietic stem cells (HSCs) remains unclear. In fact, whether human cells can be transformed as readily as murine cells by simple oncogene combinations is a subject of considerable debate. Here, we describe the development of humanized mouse model of MYC/BCL2-driven 'double-hit' lymphoma. By engrafting human HSCs transduced with the oncogene combination into immunodeficient mice, we generate a fatal B malignancy with complete penetrance. This humanized-MYC/BCL2-model (hMB) accurately recapitulates the histopathological and clinical aspects of steroid-, chemotherapy- and rituximab-resistant human 'double-hit' lymphomas that involve the MYC and BCL2 loci. Notably, this model can serve as a platform for the evaluation of antibody-based therapeutics. As a proof of principle, we used this model to show that the anti-CD52 antibody alemtuzumab effectively eliminates lymphoma cells from the spleen, liver and peripheral blood, but not from the brain. The hMB humanized mouse model underscores the synergy of MYC and BCL2 in 'double-hit' lymphomas in human patients. Additionally, our findings highlight the utility of humanized mouse models in interrogating therapeutic approaches, particularly human-specific monoclonal antibodies.

Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation

AbstractIn recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)–expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45+ cell engraftment and human CD45+CD33+ myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells.

Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheep

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.

A Novel Osteoclastic Network Determines In Vitro Niche for Mouse and Human Hematopoietic Stem Cells

Abstract 1325New innovative strategies are needed to expand human hematopoietic stem cells (HSCs) for clinical applications. In this perspective, we recently developed an in vitro/in vivo screening strategy, which revealed 18 nuclear factors that enhance HSC activity (Deneault et al., Cell 2009). During these experiments, mouse HSCs were kept for 12 days in mini-cultures that included viral producer cells for each tested factor. Interestingly, 4 of the 18 hits identified in this initial screen, i.e., Fos, Tcfec, Hmgb1 and Sfpi1 operated through non-HSC autonomous (NHA) mechanisms: expanded HSCs were not infected with retrovirus. This suggested that the transfected viral producer cells (feeder cells) produced membrane-bound or soluble molecules that promote expansion of HSCs introduced in these cultures. We now provide evidence that seven additional factors, i.e., Smarcc1, Vps72, Sox4, Klf10, Ski, Prdm16 and Erdr1 significantly enhance HSC activity through NHA mechanisms, hereafter called “NHA factors”. Moreover, we found that Vps72, Fos and Klf10 also promote expansion of human HSCs by NHA mechanisms. Interestingly at least for feeders expressing Klf10, we observed that physical contact between HSCs and these engineered support cells was not necessary, suggesting the presence of secreted molecule(s) in the medium. Expression profiling was next performed using mRNA extracted from feeder cells transduced with each of the NHA factors. Firstly, we shed light on the transcriptional make up and potential convergence of signaling pathways in the engineered feeder cells: our results reveal two independent but interconnected transcriptional regulatory subnetworks. Strikingly, some constituents of the subnetworks, i.e., Sfpi1, Fos, Klf10 and Tcfec (Mitf-related) have previously been shown to play critical roles in the regulation of osteoclasts, which are a myeloid-derived population of cells residing in the HSC bone marrow niche. In addition, all of the NHA factors act in concert to increase Prdm16 expression levels in a range from 2.5- to 54.6-fold. For this reason, Prdm16 clearly holds the position of central hub of the osteoclastic network. However, Tcfec represents the ultimate downstream effector of this pathway as Prdm16 elevates its expression levels by 18.9-fold. In parallel, Prdm16 can also increase the expression of Sfpi1, which in turn can upregulate Tcfec expression up to 874-fold. Moreover, upregulated mRNA targets corresponding to factors that are secreted or associated with the plasma membrane were considered as potential candidate agonists of HSC self-renewal. A high degree of overlap was observed between the sets of proteins produced by feeder cells engineered to overexpress Sfpi1, Fos, Klf10, Tcfec or Ski. These secreted and membrane bound proteins include Agt, Aspn, Ogn, Ptgds, Nckap1l, Rgs16 and Lcn2. Studies are ongoing to characterize the contribution of these newly identified NHA proteins in HSC expansion. Validation of these NHA proteins in human HSC expansion will have a clear potential for translational medicine. Disclosures:No relevant conflicts of interest to declare.

Small Molecule Screens for Stem Cell Expansion

Abstract SCI-42 The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening &gt;100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a &gt;600-fold increase in the number of CD34+ cells, and a &gt;2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use. Disclosures: Cooke: Novartis: Employment. Boitano:Novartis: Employment.

The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment

Substantial improvements have been made in recent years in the ability to engraft human cells and tissues into immunodeficient mice. The use of human hematopoietic stem cells (HSCs) leads to multi-lineage human hematopoiesis accompanied by production of a variety of human immune cell types. Population of murine primary and secondary lymphoid organs with human cells occurs, and long-term engraftment has been achieved. Engrafted cells are capable of producing human innate and adaptive immune responses, making these models the most physiologically relevant humanized animal models to date. New models have been successfully infected by a variety of strains of Human Immunodeficiency Virus Type 1 (HIV-1), accompanied by virus replication in lymphoid and non-lymphoid organs, including the gut-associated lymphoid tissue, the male and female reproductive tracts, and the brain. Multiple forms of virus-induced pathogenesis are present, and human T cell and antibody responses to HIV-1 are detected. These humanized mice are susceptible to a high rate of rectal and vaginal transmission of HIV-1 across an intact epithelium, indicating the potential to study vaccines and microbicides. Antiviral drugs, siRNAs, and hematopoietic stem cell gene therapy strategies have all been shown to be effective at reducing viral load and preventing or reversing helper T cell loss in humanized mice, indicating that they will serve as an important preclinical model to study new therapeutic modalities. HIV-1 has also been shown to evolve in response to selective pressures in humanized mice, thus showing that the model will be useful to study and/or predict viral evolution in response to drug or immune pressures. The purpose of this review is to summarize the findings reported to date on all new humanized mouse models (those transplanted with human HSCs) in regards to HIV-1 sexual transmission, pathogenesis, anti-HIV-1 immune responses, viral evolution, pre- and post-exposure prophylaxis, and gene therapeutic strategies.

Humanized tumor mice—A new model to study and manipulate the immune response in advanced cancer therapy

The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.

Mechanism for platelet reduction in anti-neuropilin-1 (MNRP1685A)–treated phase I patients.

e13598 Background: Neuropilin-1 (NRP1), a high affinity receptor for members of the VEGF family, is expressed on endothelial cells (EC) and tumor cells, and is involved with maturation and remodeling of blood vessels. In two phase I studies, administration of human anti-NRP1 antibody (Ab) MNRP1685A resulted in transient platelet (PLT) reductions in patients. To determine if these PLT reductions were due to PLT production or consumption, we evaluated the binding of MNRP1685A to human megakaryocyte (MK) progenitor cells and PLTs and the impact of MNRP1685A on MK development and PLT function. Methods: MNRP1685A binding to human hematopoietic stem cells (HSC), cultured MKs and PLTs was assessed by Western blot (WB), mass spectrometry and flow cytometry (FACS). MNRP1685A effects on platelets from healthy donors and patients on study were analyzed by FACS for activation markers CD62P and activated GPIIbIIIa (PAC1) and evaluated by aggregometry. MNRP1685A effects on MK development were evaluated by HALO proliferation assay and expression of CD61/41 maturation markers via FACS. To examine PLT binding to EC, fluorescein-labeled PLTs were incubated with TNF-stimulated human umbilical vein endothelial cells (HUVEC) in the presence of MNRP1685A and HUVEC-associated fluorescence was measured. Results: We demonstrate specific CD32 (FCγR2a)-dependent and independent MNRP1685A binding to human HSC, cultured MKs and PLTs by FACS and WB. There was no effect of MNRP1685A on HSC proliferation or maturation to MK. Exposure of MNRP1685A to human PLTs ex vivo or in vivo (phase 1 patients) result in modest PLT activation via a CD32-dependent mechanism as measured by FACS for CD62P and PAC1, as well as increased PLT aggregation compared to a control Ab. An increase of CD32-dependent binding of PLTs to HUVEC in vitro was observed in the presence of MNRP1685A, but not with a control Ab. Conclusions: MNRP1685A specifically binds to NRP1 on the surface of human PLTs and cells in the MK lineage, induces modest CD32-dependent PLT activation and binding to activated endothelial cells. These properties of MNRP1685A can contribute to PLT consumption and may explain decreases in circulating PLT levels in patients dosed with MNRP1685A in phase I clinical studies.

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.

Identification of HIF2α as an important STAT5 target gene in human hematopoietic stem cells

AbstractThe transcription factor signal transducer and activator of transcription 5 (STAT5) fulfills essential roles in self-renewal in mouse and human hematopoietic stem cells (HSCs), and its persistent activation contributes to leukemic transformation, although little molecular insight into the underlying mechanisms has been obtained. In the present study, we show that STAT5 can impose long-term expansion exclusively on human HSCs, not on progenitors. This was associated with an enhanced cobblestone formation under bone marrow stromal cells of STAT5-transduced HSCs. Hypoxia-induced factor 2α (HIF2α) was identified as a STAT5 target gene in HSCs, and chromatin immunoprecipitation studies revealed STAT5 binding to a site 344 base pairs upstream of the start codon of HIF2α. Lentiviral RNA interference (RNAi)–mediated down-modulation of HIF2α impaired STAT5-induced long-term expansion and HSC frequencies, whereas differentiation was not affected. Glucose uptake was elevated in STAT5-activated HSCs, and several genes associated with glucose metabolism were up-regulated by STAT5 in an HIF2α-dependent manner. Our studies indicate that pathways normally activated under hypoxia might be used by STAT5 under higher oxygen conditions to maintain and/or impose HSC self-renewal properties.

Mesenchymal Stem Cells Secreting Angiopoietin-Like-5 Support Efficient Expansion of Human Hematopoietic Stem Cells Without Compromising Their Repopulating Potential

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.

Modeling Human T Cell Acute Lymphoblastic Leukemia In Vitro and In Vivo with LMO2.

AbstractAbstract 3648The studies identifying gene translocations and mutations in T-ALL cell lines and/or in patients have contributed significantly to the understanding of the genetic abnormalities involved in T-ALL. However, studies on the biology of these genes, the targeted cells, the sequence and the number of hits required to convert a primary human hematopoietic stem cell (HSC)/progenitor cell into a fully transformed leukemic cell require good experimental models of human T cell development both in vivo and in vitro. The only in vivo model of human T cell leukemogenesis came unexpectedly from the gene therapy trial on patients with X-linked severe combined immunodeficiency (SCID-X1). Three to five years after gene therapy, 4 out of 10 patients in the trial developed clonal T-ALL. In these patients, retroviral integrations were found in proximity to the LMO2 promoter in the malignant clones, leading to aberrant expression of the oncogene. However, little is known on the effect of LMO2 overexpression in human cells and how it facilitates the development of T-ALL.We have developed in vivo and in vitro models to study the role of T cell oncogenes in human cells. Using the OP9-DL1 co-culture system to differentiate human HSC into mature T cells in vitro, we culture human HSC transduced with lentiviruses expressing LMO2. LMO2 overexpressing cells are blocked at the double negative stage (CD4-CD8-) of differentiation when co-cultured on OP9-Delta-Like1 stroma and proliferate 50 to 100 times more than control cells. However, these cells are not immortalized and cultures lasted approximately 80 days. LMO2 overexpression have no effect on myeloid differentiation in vitro.In vivo, LMO2 transduced human HSC/progenitor cells engraft the bone marrow of immunodeficient mice to levels comparable to control cells, while normal myeloid and B cell populations 20–24 weeks post-transplantation. LMO2 transduced cells have an increased capacity to generate T cells in the thymus in comparison to control cells (42% engraftment vs 8%, p<0.0001). Surprisingly, thymic and peripheral LMO2 cells are not blocked in their differentiation. LMO2 cells did not engraft secondary mice, confirming that LMO2 doesn't induce self-renewal of human HSC. However, the increase in thymic repopulation by LMO2 cells and the lack of differentiation block in vivo suggest that LMO2 overexpression generates an abnormal T cell population with an increase repopulation advantage (increase proliferation or decrease apoptosis) in the thymus which becomes the substrate for additional genetic/epigenetic events.To test this hypothesis, we tried to immortalize LMO2 cells in vitro with secondary hits. Our preliminary results show that insertional mutagenesis can immortalized LMO2 cells in vitro. However these cells are not able to engraft immunodeficient mice or generate leukemia in vivo. The addition of intracellular NOTCH to one immortalized LMO2 cell line allows these cells to engraft and generate human T-ALL in vivo.Globally, these results show that T cell oncogenes can be studied in primary human hematopoietic cells both in vitro and in vivo. Also, at least three hits are required to transform a human primary HSC/progenitor cell into a leukemic cell able to engraft and generate leukemia in vivo. It also suggests that a non-engrafting cell can be turned into a leukemic cell generating leukemia in vivo, implying that a cell can regain self-renewing properties. Disclosures:No relevant conflicts of interest to declare.