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Objective: To compare the macular area parameters and aqueous humor factors between myopia and emmetropia. Methods: Convenience sampling was used to select patients who visited the Changzhi Aier Eye Hospital's department of ophthalmology from December 2018 to December 2022 as the study participants. They were divided into three groups according to whether they were diagnosed as mild myopia myopic, highly myopic or not as follows: the mild myopia group (60 cases, 108 eyes), the high myopia group (46 cases, 78 eyes) and the healthy emmetropia group (40 cases, 65 eyes). The differences in the macular integrity (MI) assessment, optical coherence tomography and optical coherence tomography angiography parameters and aqueous humor factors were compared between the three groups. Results: AL in high myopia group was the highest, and that in emmetropia group was the lowest. The BCVA of mild myopia group was the highest. The RS in the high myopia group were significantly lowest in the three groups (26.42 ± 1.04 vs. 28.34 ± 0.76 vs. 31.92 ± 0.77) (F = 5.374, p = 0.013). The 63% BCEA, 95% BCEA and MI in the high myopia group were significantly highest (p < 0.05). The mean RPE thickness, mean CT and mean RT in the high myopia group were lowest (p < 0.05). The blood flow density were lowest in the superficial fovea, paracentral fovea and different subdivisions of the paracentral fovea in the high myopia group (p < 0.05). The VEGF concentration in the aqueous humor of the high myopia group was lowest (25.62 ± 17.43 vs. 32.45 ± 24.67 vs. 64.37 ± 21.14) (F = 9.237, p < 0.001). The MMP-2 concentration was highest (483 ± 201.48 vs. 410 ± 142.37 vs. 386 ± 154.34) (F = 5.542, p = 0.018). The VEGF concentration in the aqueous humor factor was negatively correlated with the AL in the myopia group (r = -0.438, p = 0.002), the MMP-2 concentration was positively correlated with the AL (r = 0.484, p = 0.010). Conclusion: Patients with high myopia showed decreased retinal light sensitivity, fixation stability, superficial blood flow density and retinal thickness compared with people with emmetropia. A decreased VEGF concentration and increased MMP-2 concentration in the aqueous humor factor have potential associations with the development of high myopia.

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p15INK4b (cyclin-dependent kinase inhibitor 2B, CDKN2B, p15), a cyclin-dependent kinase inhibitor (CKI) belonging to the INK4 family, plays an important role in hematopoiesis. Its expression level was positively related to the blockage effects of RUNX1b at the early stage. Experiments using human embryonic stem cell (hESC) lines with inducible p15 expression suggested that p15 overexpression can significantly decrease the proportion of KDR+ cells in S and G2-M stages 4 days after induction from day 0. Moreover, p15 overexpression from the early stage can decrease production of CD34highCD43- cells and their derivative populations, but not CD34lowCD43- cells. These effects were weakened if induction was delayed and disappeared if induction started after day 6. All these effects were counteracted by inhibition of TGF-ß signaling. TGF-ß1 stimulation elicited similar effects as p15 overexpression. RUNX1 overexpression and activation of the TGF-ß signaling pathway upregulate the expression of p15, which is partially responsible for blockade of hematopoiesis and relevant to a change in the cell cycle status. However, it is possible that other mechanisms are involved in the regulation of hematopoiesis.

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The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1-#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3-4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-ß) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.

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Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43- cells and derivative populations, but not that of CD34lowCD43- cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-ß or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

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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.

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BACKGROUND AND OBJECTIVES: p21, an important member of the Cip/Kip family, is involved in inhibitory effects of RUNX1b overexpression during the early stage of human hematopoiesis. METHODS AND RESULTS: We established a human embryonic stem cell (hESC) line with inducible expression of p21 (p21/hESCs). Overexpression of p21 did not influence either mesoderm induction or emergence of CD34＋ cells, but it significantly decreased the production of CD43＋ cells and changed the expression profile of hematopoiesis-related factors, leading to the negative effects of p21 on hematopoiesis. CONCLUSIONS: In RUNX1b/hESC co-cultures when RUNX1b was induced from D0, perturbation of the cell cycle caused by upregulation of p21 probably prevented the appearance of CD43＋ cells, but not CD34＋ cells. The mechanisms via which CD34＋ cells are blocked by RUNX1b overexpression remain to be elucidated.

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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.

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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.

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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.

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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.

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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.

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The development of human erythroid cells has been mostly examined in models of adult hematopoiesis, while their early derivation during embryonic and fetal stages is largely unknown. We observed the development and maturation of erythroblasts derived from human pluripotent stem cells (hPSCs) by an efficient co-culture system. These hPSC-derived early erythroblasts initially showed definitive characteristics with a glycophorin A+ (GPA+) CD34lowCD36- phenotype and were distinct from adult CD34+ cell-derived ones. After losing CD34 expression, early GPA+CD36- erythroblasts matured into GPA+CD36low/+ stage as the latter expressed higher levels of ß-globin along with a gradual loss of mesodermal and endothelial properties, and terminally suppressed CD36. We establish a unique in vitro model to trace the early development of hPSC-derived erythroblasts by serial expression of CD34, GPA, and CD36. Our findings may provide insight into the understanding of human early erythropoiesis and, ultimately, therapeutic potential.

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