Overexpression of HOXA9 upregulates NF-κB signaling to promote human hematopoiesis and alter the hematopoietic differentiation potentials.

BACKGROUND: The HOX genes are master regulators of embryogenesis that are also involved in hematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles in hematopoiesis and leukemogenesis. METHODS: We established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normal pluripotency and potential for hematopoiesis, which could be used to analyze gene function with high accuracy. HOXA9/hESCs co-cultured with aorta-gonad-mesonephros-derived stromal cells (AGM-S3) were induced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started and then subjected to flow cytometry. RESULTS: Induction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increased the production of CD34+ cells and derived populations. The potential for myelogenesis was significantly elevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion of S phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κB signaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could be counteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX. CONCLUSIONS: Overexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promoted hematopoiesis and the production of myeloid progenitors while reduced the production of erythroid progenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoietic lineages.

Early development and functional properties of tryptase/chymase double-positive mast cells from human pluripotent stem cells.

Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other connective tissues and expresses both tryptase and chymase, while the second, termed MCT, which only expresses tryptase, is found primarily in the mucosa. MCs induced from human adult-type CD34+ cells are reported to be of the MCT type, but the development of MCs during embryonic/fetal stages is largely unknown. Using an efficient coculture system, we identified that a CD34+c-kit+ cell population, which appeared prior to the emergence of CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), stimulated robust production of pure Tryptase+Chymase+ MCs (MCTCs). Single-cell analysis revealed dual development directions of CD34+c-kit+ progenitors, with one lineage developing into erythro-myeloid progenitors (EMP) and the other lineage developing into HSPC. Interestingly, MCTCs derived from early CD34+c-kit+ cells exhibited strong histamine release and immune response functions. Particularly, robust release of IL-17 suggested that these early developing tissue-type MCTCs could play a central role in tumor immunity. These findings could help elucidate the mechanisms controlling early development of MCTCs and have significant therapeutic implications.

Alpha lipoic acid promotes development of hematopoietic progenitors derived from human embryonic stem cells by antagonizing ROS signals.

Antagonism of ROS signaling can inhibit cell apoptosis and autophagy, thus favoring the maintenance and expansion of hematopoietic stem cells. Alpha lipoic acid (ALA), a small antioxidant molecule, affects cell apoptosis by lowering the ROS level. In this study, we show that ALA promoted production of human pluripotent stem cells (hPSCs) derived hemogenic endothelial cells and hematopoietic stem/progenitor cells in vitro. Transcriptome analysis of hPSCs derived hemogenic endothelial cells showed that ALA promoted endothelial-to-hematopoietic transition by up-regulating RUNX1, GFI1, GFI1B, MEIS2, and HIF1A and down-regulating SOX17, TGFB1, TGFB2, TGFB3, TGFBR1, and TGFBR2. ALA also up-regulated sensor genes of ROS signals, including HIF1A, FOXO1, FOXO3, ATM, PETEN, SIRT1, and SIRT3, during the process of hPSCs derived hemogenic endothelial cells generation. However, in more mature hPSC-derived hematopoietic stem/progenitor cells, ALA reduced ROS levels and inhibited apoptosis. In particular, ALA enhanced development of hPSCs derived hematopoietic stem/progenitor cells by up-regulating HIF1A in response to a hypoxic environment. Furthermore, addition of ALA in ex vivo culture greatly improved the maintenance of functional cord blood HSCs by in vivo transplantation assay. Our findings support the conjecture that ALA plays an important role in efficient regeneration of hematopoietic stem/progenitor cells from hPSCs and maintenance of functional HSCs, providing insight into understanding of regeneration of early hematopoiesis for engineering clinically useful hPSCs derived hematopoietic stem/progenitor cells transplantation. Thus, ALA can be used in the study of hPSCs derived HSCs.

RUNX1-205, a novel splice variant of the human RUNX1 gene, has blockage effect on mesoderm-hemogenesis transition and promotion effect during the late stage of hematopoiesis.

Runt-related transcription factor 1 (RUNX1) is required for definitive hematopoiesis; however, the functions of most human RUNX1 isoforms are unclear. In particular, the effects of RUNX1-205 (a novel splice variant that lacks exon 6 in comparison with RUNX1b) on human hematopoiesis are not clear. In this study, a human embryonic stem cell (hESC) line with inducible RUNX1-205 overexpression was established. Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+ cells, and the production of hematopoietic stem/progenitor cells was significantly reduced. In addition, the expression of hematopoiesis-related factors was downregulated. However, these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells, indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells, while the promotive effect could be observed during the late stage of hematopoiesis. This is very similar to that of RUNX1b. Interestingly, the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b, and the protein stability of RUNX1-205 was much higher than that of RUNX1b. Thus, the function of RUNX1-205 in normal and diseased models should be further explored.

HOXC4 up-regulates NF-κB signaling and promotes the cell proliferation to drive development of human hematopoiesis, especially CD43+ cells.

The hematopoietic function of HOXC4 has not been extensively investigated. Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells. CD34-CD43+ cells could be clearly classified into CD34-CD43low and CD34-CD43high sub-populations at D14. The former cells had greater myelogenic potential, and their production was not significantly influenced by induction of HOXC4. By contrast, the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells, and thus had properties of erythroid-megakaryocyte common progenitors, which abundance was increased by ∼2-fold when HOXC4 was induced from D10. For CD34-CD43low, CD34+CD43+, and CD34-CD43high sub-populations, CD43 level served as a natural index for the tendency to undergo hematopoiesis. Induction of HOXC4 from D10 caused more CD43+ cells sustain in S-phase with up-regulation of NF-κB signaling, which could be counteracted by inhibition of NF-κB signaling. These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status, which containing potential of clinical applications.

The piggyBac-based double-inducible binary vector system: A novel universal platform for studying gene functions and interactions.

Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293 T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.

Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors.

GATA2 is essential for the endothelial-to-hematopoietic transition (EHT) and generation of hematopoietic stem cells (HSCs). It is poorly understood how GATA2 controls the development of human pluripotent stem cell (hPSC)-derived HS-like cells. Here, using human embryonic stem cells (hESCs) in which GATA2 overexpression was induced by doxycycline (Dox), we elucidated the dual functions of GATA2 in definitive hematopoiesis before and after the emergence of CD34+CD45+CD90+CD38- HS-like cells. Specifically, GATA2 promoted expansion of hemogenic precursors via the EHT and then helped to maintain HS-like cells in a quiescent state by regulating cell cycle. RNA sequencing showed that hPSC-derived HS-like cells were very similar to human fetal liver-derived HSCs. Our findings will help to elucidate the mechanism that controls the early stages of human definitive hematopoiesis and may help to develop a strategy to generate hPSC-derived HSCs.

Establishment of an in vitro system based on AGM-S3 co-culture for screening traditional herbal medicines that stimulate hematopoiesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Spatholobus suberectus Dunn is a traditional Chinese medicine (TCM) that can activate blood, dispel stasis, inhibit platelet aggregation, and stimulate hematopoiesis, and thereby treat anemia and diseases related to blood stasis syndrome (BSS). However, its hematopoiesis-stimulating activity is not well understood. AIM OF STUDY: Four phenolic compounds (daidzein, formononetin, catechin, and procyandin B2) were isolated and purified from stems of S. suberectus, and tested using an in vitro hematopoiesis system. MATERIALS AND METHODS: An AGM-S3 co-culture system for hematopoiesis derived from human embryonic stem cells (hESCs) was employed to explore effects on hematopoiesis. At different stages, extracts from Spatholobus suberectus Dunn were added to the co-culture system at concentrations of 2, 10, or 50 µM, and fluorescence-activated cell sorting (FACS), hematopoietic colony culturing, and quantitative reverse transcription PCR (qRT-PCR) were used to probe changes in hematopoietic progenitors and erythroid progenitors. RESULTS: When H1 hESCs co-cultured with AGM-S3 were added along with 10 µM catechin from day 12 (D12), proliferation and differentiation of hematopoietic and erythroid progenitors from hESCs was increased based on FACS with antibodies recognizing CD34/CD45 and GPA/CD71. Hematopoiesis colony culturing further confirmed the promotion effect of catechin on hematopoiesis, and other active fractions did not significantly promote hematopoiesis. qRT-PCR revealed that some important genes related to hematopoiesis and erythroid were up-regulated followed catechin exposure. CONCLUSIONS: Our results demonstrate that catechin, an active ingredient of Spatholobus suberectus Dunn, can increase the efficiency of hematopoiesis, including hematopoietic and erythroid progenitors, consistent with previous reports. The AGM-S3 co-culture system could provide an effective tool for screening active compounds in TCMs that promote hematopoiesis, and may be of clinical and pharmaceutical use.

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-ß signaling pathway, indicating a close relationship between RUNX1b/c and TGF-ß pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models.

MicroRNA-17 post-transcriptionally regulates polycystic kidney disease-2 gene and promotes cell proliferation.

To identify the possible microRNAs (miRNAs) which target the polycystic kidney disease-2 gene (PKD2), and clarify effects of the miRNAs on PKD2. We preliminarily used bioinformatics to analyze 3'UTR (3'untranslated regions) of PKD1 and PKD2 in order to predict the potential microRNAs targeted on them. Subsequently, the stable cell lines with overexpression of microRNA-17 (miR-17) were screened, and luciferase assay combined with the mutation 3'UTR of PKD2 were performed to verify PKD2 is the target of miR-17. Moreover, RT-PCR and Western Blotting were used to determine the post-transcriptionally regulation of PKD2 by miR-17. Finally, MTT cell assays allied with PKD2 rescued strategy were employed to evaluate cell proliferation effects. Our study firstly found that the 3'UTR of PKD2 was more conservation than that of PKD1, and microRNA-17 directly targets the 3'UTR of PKD2 and post-transcriptionally repress the expression of PKD2. Moreover, our findings also demonstrated that overexpression of miR-17 may promote cell proliferation via post-transcriptionally repression of PKD2 in HEK 293T. This suggested that microRNA might be a novel mechanism for cystogenesis as well as a potential therapeutic target for the cell proliferation of autosomal dominant polycystic kidney disease (ADPKD).

Down-regulation of Pkd2 by siRNAs suppresses cell-cell adhesion in the mouse melanoma cells.

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell-cell or cell-matrix adhesion, suggesting that PC-2 may play a role in cell-cell/cell-matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell-cell adhesion, but not cell-matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell-cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell-cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell-cell adhesion, at least partially, through E-cadherin.

TC1 (C8orf4) is involved in ERK1/2 pathway-regulated G(1)- to S-phase transition.

Although previous studies have implicated a role for TC1 (C8orf4) in cancer cell proliferation, the molecular mechanism of its action is still largely unclear. In this study, we showed, for the first time, that the mRNA levels of TC1 were upregulated by mitogens (FBS/thrombin) and at least partially, through the ERK1/2 signaling pathway. Interestingly, the over-expression of TC1 promoted the G(1)- to S-phase transition of the cell cycle, which was delayed by the deficiency of ERK1/2 signaling in fibroblast cells. Furthermore, the luciferase reporter assay indicated that the over-expression of TC1 significantly increased Cyclin D1 promoter-driven luciferase activity. Taken together, our findings revealed that TC1 was involved in the mitogen-activated ERK1/2 signaling pathway and positively regulated G(1)- to S-phase transition of the cell cycle. Our results may provide a novel mechanism of the role of TC1 in the regulation of cell proliferation.