Developmental Pluripotency Associated 5 (Dppa5) Regulates Hematopoietic Stem Cell Reconstitution Capacity by Modulating Cellular Metabolism and ER Stress

Abstract 847

Expansion of hematopoietic stem cells (HSCs) will be required to achieve safe and effective transplantation for treatment of malignant blood disorders in adults. Due to regulatory constraints in the bone marrow (BM) stem cell niche, HSCs are quiescent under steady state conditions. Identification of regulators with a potential to expand HSC requires model systems with dividing stem cells, for example embryonic stem (ES) cells. To discover novel regulators that can increase the number of HSCs, we designed a screening protocol to identify possible candidates by comparing gene expression in undifferentiated and differentiated ES cells. We developed several key criteria to select candidates from microarray data and identified four extrinsic soluble factor candidates. One of these candidates, Cripto, was characterized as a critical regulator of HSC in the endosteal hypoxic niche by inducing higher glycolytic activity as an intermediary of the master regulator of hypoxic responses, HIF-1α (Miharada et al, Cell Stem Cell, 2011).

Same strategy identified ten intrinsic factors. After initial selection, a second screening of the selected genes using a lentiviral over-expression approach in CD34⁻CD48⁻KSL cells identified three potential candidate genes. One of these candidates, Dppa5 (developmental pluripotency associated 5), generated more than 100-fold expansion of phenotypic HSCs upon enforced expression after two weeks culture in vitro. Overexpression of Dppa5 robustly increased the reconstitution level in lethally irradiated mice (15-fold higher than control, n=7, p<0.001) without loosing multi-lineage potential after 14 days culture in vitro, while this enhancement was not seen in the short-term (2 days) culture. It was noteworthy that the enhanced reconstitution by Dppa5 was significantly increased in the secondary recipients, whereas control vector transduced cells failed to reconstitute (n=14, p<0.001).

Quantitative real time PCR (qRT-PCR) analysis revealed that the expression of Dppa5 mRNA is significantly higher in long-term HSCs than other progenitor populations. Conversely, down-regulation of Dppa5 using shRNA resulted in a significant severe reduction of reconstitution of the transplanted Dppa5 knockdown HSCs (12-fold reduction, n=6, p<0.001).

In order to study what factors are affected by Dppa5 overexpression, two-dimensional gel electrophoresis (2D-DIGE) was performed. The findings showed that the levels of proteins involved in DNA synthesis and protein translation were increased. In contrast, decreased proteins included glycolysis related enzymes and ER stress response proteins. Surprisingly, all decreased glycolysis proteins (pyruvate kinase M2 (Pkm2), phosphoglycerate kinase 1 (Pgk1), and protease serine 1) have been observed as increased proteins when hematopoietic cells were cultured with recombinant Cripto. These proteins, as well as Cripto, are regulated by HIF-1α. Importantly, loss of Pkm2 has been shown to lead to a competitive repopulative advantage following bone marrow transplantation by modulating HSC metabolism (Wang et al., Abstract ISSCR 2012). These reported findings are consistent with our results. Moreover, GRP78 (a cell surface receptor for Cripto), GRP94, Calreticulin, and Pdia3 were decreased upon Dppa5 overexpression: These molecules are key components of ER stress chaperones and also known to be under the control of HIF-1α. Since these findings strongly indicate alteration in HSC metabolism, we measured cellular mitochondrial activity. MitoTracker (MT) staining showed higher intensity of MT in the Dppa5 over-expressed HSC population in the engrafted mice compared with the control (1.36-fold increase, n=4, p<0.05) suggesting that oxidative phosphorylation is enhanced.

Molecular analysis has demonstrated that Dppa5 binds to multiple target RNA sequences and regulates the expression of several targets. UV-crosslinking immunoprecipitation (UV-CLIP) followed by qRT-PCR demonstrated binding of Dppa5 to the RNA sequence of GRP94 in hematopoietic cell lines overexpressing Dppa5, in addition to the previously reported genes (Mapk6, Cdc42, and Cyclin F). Thus, Dppa5 is a novel critical regulator of HSC and our preliminary findings indicate that its mechanism is governed by metabolism switching and decreased ER stress responses through GRP94 as an important bridging factor.