Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the ex vivo expansion and maintenance of hematopoietic stem/progenitor cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported. METHODS: Flow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs. RESULTS: The stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation. CONCLUSIONS: A novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell-cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

RUNX1 overexpression triggers TGF-ß signaling to upregulate p15 and thereby blocks early hematopoiesis by inducing cell cycle arrest.

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.

Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythroblasts.

The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation. RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes, favoring their maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-ß and NF-κB signaling.

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).

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.

Overexpression of p21 Has Inhibitory Effect on Human Hematopoiesis by Blocking Generation of CD43＋ Cells via Cell-Cycle Regulation.

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.

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.

Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a, CD34, and CD36.

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.

Nuclear factor kappa B signaling initiates early differentiation of neural stem cells.

Inflammatory mediators, many of which activate the signaling of nuclear factor kappa B (NFκB), have received increasing attention in the field of neurogenesis. NFκB signaling regulates neurite outgrowth and neural plasticity as well as the proliferation/apoptosis and terminal differentiation of neural stem cells (NSCs). Early neurogenesis from NSCs produces identical progeny through symmetric division and committed daughter cells through asymmetric division. Here, we show that NFκB signaling is required for NSC initial differentiation. The canonical IKKß/IκBα/p65 pathway is activated during the initial stages of neural differentiation induced by treatment with TNFα or withdrawal of epidermal growth factor/basic fibroblast growth factor. NSC-specific inhibition of NFκB in transgenic mice causes an accumulation of Nestin(+) /Sox2(+) /glial fibrillary acidic protein(+) NSCs. Inhibition of NFκB signaling in vitro blocks differentiation and asymmetric division and maintains NSCs in an undifferentiated state. The induction of initial differentiation and asymmetry by NFκB signaling occurs through the inhibition of C/EBPß expression. Our data reveal a novel function of NFκB signaling in early neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases.