ALBERT

Description: Abstract 562 Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV infected patients. In this study, we have utilized human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were de-differentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs towards the hematopoietic lineage, a robust quantity (〉35%) of colony forming CD133+ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection (〉3 logs) from HIV-1 infection. Here we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies. Disclosures: No relevant conflicts of interest to declare.

Description: Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow–transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit+/lineage−/Sca-1+ (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.

Description: Abstract 1451 Poster Board I-474 Mesenchymal stem cells (MSC, aka marrow stromal cells) present a promising tool for cell therapy, and have been shown to contribute to the recovery of tissues in myocardial infarction, stroke, meniscus injury, limb ischemia, and even neurodegenerative disorders. The percentage of engrafted MSC in these studies has been very low in comparison to the recipient tissue cells, suggesting that their efficacy relies upon actions other than differentiation. One theory of tissue repair and regeneration by adult MSC is that the injected stem cells home to the injured area, in particular to hypoxic, apoptotic, or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive products can suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, proliferation and differentiation of tissue-residing stem cells. Paracrine effects exerted by MSC are distinct from the classical model of direct differentiation of stem cells into the tissue to be regenerated. MSC can, however, directly contribute to the repair of bone, tendon and cartilage. In some cases, where the patient lacks expression of a critical gene product, genetic engineering of the MSC is desired. Using human embryonic stem cells (hESC), the integration site of a vector can be fully characterized and the clones with benign integration sites can be expanded. Homologous recombination is also now feasible for embryonic cells due to increased efficiencies, and clones with vectors targeted for gene correction can be expanded. For this reason we have generated MSC from the well-characterized human embryonic stem cell line H9. H9-derived MSC (H9-MSC) expressed CD105, CD90, CD73 and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR. H9-MSC also lacked expression of the hESC pluripotency markers SSEA-4 and Tra-1-81, which were expressed by the starting H9 line. Additionally, they lacked expression of SSEA-1, an early marker of hESC differentiation. Marrow-derived MSC showed a similar phenotype when analyzed concurrently with H9-MSC. Morphology was similar to adult MSC derived from marrow or adipose tissue. H9-MSC grew more robustly than MSC derived from marrow or adipose tissue, with a rapid doubling time and a failure to display contact inhibition. However, upon reaching maturity the cells did slow to the same rate as bone marrow-derived MSC, and then were finally subject to contact inhibition. Additionally, H9-MSC were injected with matrigel subcutaneously into the flank of immune deficient mice [NOD/SCID/IL2Rg-/- (NSG)] to assess their ability to form tumors due to possible growth abnormalities. No mice that received injections of H9-MSC formed teratoma or other tumors, whereas the parent H9 line robustly forms teratoma. H9-MSC could be robustly differentiated into bone, as shown by alizarin red staining of mineralized plaques after 21 days of culture in standard osteogenic differentiation medium containing 0.2 mM ascorbic acid, 0.1 m M dexamethasone, and 10 mM b-glycerophosphate with media changes every three days. H9-MSC morphology was noticeably different from that of H9-MSC undergoing differentiation as early as day 3 of the differentiation protocol. Finally, cells were tested for their capacity to respond to a hypoxic in vivo environment, using our standard hindlimb ischemia model in immune deficient [NOD/SCID/b2M-/-] mice. H9-MSC were found to have homed to the hypoxic muscle by 48 hours after injection into the bloodstream. In summary we have defined methods for differentiation of hESC into MSC, have defined their characteristics, and in vivo migratory properties. This system could be useful, following further safety studies, for production of large numbers of MSC from embryonic or induced pluripotent stem cells that have been corrected for gene defects by lentiviral vector integration with careful assessment of integration site, or by homologous integration, with subsequent expansion, characterization and banking of the line prior to differentiation into functional mesenchymal stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 946 Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that haploinsufficiency of del(5q) genes maybe an early genetic event. To test whether haploinsufficiency of del(5q31.2) genes contribute to ineffective hematopoiesis observed in MDS (i.e. abnormal proliferation, apoptosis, and differentiation), we reduced the expression of HSPA9 (Hspa9 for the murine orhologue), one of the 28 genes, in primary human hematopoietic cells and a murine transplantation model using lentiviral mediated gene silencing. We purified human CD34+ hematopoietic progenitors from cord blood samples (〉90% purity) and infected them individually with 5 unique lentiviral shRNAs targeting HSPA9 (each achieving 30% to 〉90% knockdown) and 2 control shRNAs. HSPA9 knockdown cells expanded an average of 6 fold less than control cells from days 4-7 in erythroid unileage differentiation culture conditions (SCF, IL-6, IL-3, and EPO) (p≤0.007). The reduced cell numbers observed in HSPA9 knockdown cultures was associated with an increase in apoptosis and a decrease in the number of cells in S-phase compared to control shRNA expressing cells (p≤0.008) (increased membrane depolarization and active Caspase-3 expression were also observed with HSPA9 knockdown). In addition, after 7 days, there was a significant reduction in maturing erythroid precursors (p≤0.004), but not myeloid or megakaryocytic precursors, in HSPA9 knockdown cultures vs. control cultures. These results suggest that reduced expression of HSPA9 in primary hematopoietic cells recapitulates key features of ineffective erythropoiesis observed in MDS. We extended these studies using a bone marrow lentiviral transduction/transplantation mouse model. Bone marrow progenitors were infected with a shRNA expressing lentivirus with yellow fluorescence protein (YFP) as a reporter. Loss of Hspa9 shRNA transduced peripheral blood cells occurred over 4-months, necessitating evaluation of mice from 3-8 weeks post-transplant. Two independent transplantation cohorts were performed and analyzed (control shRNA, n=8 mice; Hspa9 shRNA, n=9 mice). The percent of YFP donor leukocytes was significantly reduced in bone marrow (36.3% vs. 27.9%, p≤0.02), spleen (41.6% vs. 19.5%, p≤0.001), and peripheral blood (39.5% vs. 29.5%, p≤0.007) in mice receiving Hspa9 knockdown cells, consistent with in vitro results. The absolute number of YFP+, Ter119High/CD71+ erythroid cells was reduced by 2.5 fold in the bone marrow (2.21×106 vs. 8.97×105, p≤0.001), 8.9 fold in the spleen (3.46×106 vs. 3.87×105, p≤0.002), and 4.4 fold in the peripheral blood (20.9×103/μl vs. 4.80×103/μl, p≤0.01), whereas the total number of myeloid cells (CD11b+/Gr-1− monocytes and CD11b+/Gr-1+ neutrophils) in the three compartments was no different in mice receiving control vs. Hspa9 knockdown cells, respectively. Furthermore, the absolute number of reticulocytes, polychromatic normoblasts, and basophillic normoblasts (defined by flow cytometry) in Hspa9 knockdown recipients were significantly reduced in the bone marrow, spleen, and peripheral blood vs. control (p≤0.01 for all comparisons). The absolute number of B-lymphocytes (B220+), but not T-lymphocytes (CD3e+), were reduced by 2.4 fold in the bone marrow (1.16×107 vs. 4.81×106, p≤0.001), 3.4 fold in the spleen (4.26×108 vs. 1.24×108, p≤0.001), and 3.5 fold in the peripheral blood (1.06×103/μl vs. 3.05×102/μl p≤0.004). These results suggest that erythroid and B-cell progenitors are preferentially affected by Hspa9 knockdown, both findings that are also observed in patients with MDS. Following a 24-hour BrdU labeling in vivo, the percent of YFP+ cells in S-phase was significantly reduced in Hspa9 knockdown cells vs. control cells (21.3% vs. 27.2%, respectively, p≤0.02) in the bone marrow. Furthermore, the absolute number of YFP+ progenitor cells (lineage−/Sca-1+/C-kit+) in the bone marrow was reduced by 3.9 fold (2.59×104 vs. 6.59×103, p≤0.03), indicating that Hspa9 knockdown alters the early progenitor pool. Collectively, these results suggest that haploinsufficiency of HSPA9 alone contributes to ineffective erythropoiesis, but that other cooperating genes are necessary for clonal dominance to occur in MDS. Because HSPA9 has been shown to physically bind and sequester p53 in the cytoplasm, we are testing whether the apoptosis and cell cycle abnormalities are p53 dependent using p53-/- donor murine bone marrow cells in a transduction/transplantation model. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis. Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that del(5q) maybe an early genetic event. We hypothesize that haploinsufficiency of one or more of the 28 candidate genes in the 5q31.2 commonly deleted segment (CDS) are important for MDS pathogenesis. Many genes in the CDS, including HSPA9B, have dose reduced mRNA levels in CD34+ cells from MDS patients with del(5q), and zebrafish carrying a heterozygous mutation in the orthologue of the human HSPA9B gene display increased apoptosis in blood cells. Therefore, we asked whether reduced HSPA9B expression in primary human hematopoietic cells can recapitulate key features of ineffective hematopoiesis (i.e. abnormal proliferation, apoptosis, and differentiation). To address this question, we purified human CD34+ hematopoietic progenitors from cord blood samples (〉90% purity) and infected them with a shRNA expressing lentivirus that also carries the puromycin resistance gene. Cell culture densities were normalized after 4 days in selection medium, and 3 days later apoptosis (AnnexinV/7-AAD), cell cycle status (BrdU/7-AAD), and erythroid or myeloid differentiation was measured using flow cytometry. We performed 4–5 independent experiments using 5 individual shRNAs targeting HSPA9B (30% to 〉90% knockdown) and 2 control shRNAs. Cells transduced with control shRNAs expand 18.2 fold from days 4–7 in RBC unilineage differentiation culture medium (25ng/ml SCF, 10ng/ml IL-6, 10ng/ml IL-3 and 0.5U/ml Epo), vs. only a 0.8–5.2 fold expansion when HSPA9B is knocked down (N=5 for each shRNA, p≤0.007). The reduced cell numbers observed in HSPA9B knockdown cultures is associated with an increase in apoptosis and a decrease in the number of cells entering S-phase compared to control shRNA expressing cells. 8.2% of cells in control cultures were AnnexinV+/7AAD+ vs. 20–62% of cells in HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.0004). Following 1 hour of BrdU exposure, 60% of cells in control cultures were in S-phase vs. only 22–48% of HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.008). In addition, after 7 days in RBC unilineage differentiation culture medium, 58% of control shRNA expressing cells were CD71+/Gycophorin A+ vs. 7.1–32% of cells in the HSPA9B knockdown cultures (N=4 for each shRNA, p≤0.004). The decreased number of GPA+ cells in HSPA9B knockdown cultures was concomitant with retention of CD34+ expression on cells. Similar results were observed using myeloid unilineage culture conditions (10ng/ml SCF, 100ng/ml G-CSF, 20% FCS), where the number of cells expressing CD15+ was reduced from 47% in control cultures to 28–37% in HSPA9B knockdown cultures. A delay in erythroid and myeloid differentiation was confirmed by cell morphology. Lentiviral knockdown of the murine orthologue of HSPA9B (Hspa9a) in 2 independent transduction/transplantation experiments resulted in a loss of transduced cells over 2 months (2 shRNAs each) compared to a control shRNA, implicating a cell intrinsic defect when Hspa9a levels are reduced in vivo. Collectively, these results implicate that reduced HSPA9B expression in human CD34+ progenitor cells results in abnormal proliferation, increased apoptosis, and altered differentiation, key features of ineffective hematopoiesis, in a dose dependent manner.