Search

Background: Human hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic organoids is an unaddressed challenge because of the complexity of hematopoietic tissues., Methods: To manufacture hematopoietic organoids, we obtained CD34 + hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34 + HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids., Results: The GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation., Conclusions: These findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing., (© 2024. The Author(s).)

'Requirements for human haematopoietic stem/progenitor cells' is the first set of guidelines on human haematopoietic stem/progenitor cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, inspection methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements for human haematopoietic stem/progenitor cells, which is applicable to the quality control for human haematopoietic stem/progenitor cells. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human haematopoietic stem/progenitor cells for applications., (© 2021 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Trillium tschonoskii is a medicinal plant known to biosynthesize steroidal saponins. A phytochemical investigation of the rhizomes of T. tschonoskii led to the isolation of nine new furostanol saponins ( 1 - 9 ) and 11 known analogues ( 10 - 20 ). Five of these new compounds were shown to have hydroxy groups at the C-5 and C-6 positions, while two possess a rare aglycone containing carbonyl groups at the C-16 and C-22 positions as well as a Δ 17(20) double bond, and the others have conjugated double bonds in the E-ring or have different sugar chains at the C-3 position. All the isolates were tested for their effect on the expansion of human cord blood (CB) CD34 + hematopoietic stem and progenitor cells. It was found that CB CD34 + cells treated with compounds 6 , 7 , 9 , 10 , 14 , 15 , and 19 showed increased numbers of rigorously phenotype-defined hematopoietic stem cells. Notably, compounds 9 , 10 , 13 , and 14 demonstrated an enhanced ability to increase the percentages and numbers of CB CD34 + CD38 - cells and multipotential progenitors. The present study is the first to report that furostanol saponins from T. tschonoskii rhizomes can promote hematopoietic stem/progenitor cell (HSPC) expansion.

We reported a novel function of recombinant human thrombopoietin (TPO) in increasing hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM). Single doses of TPO treatment to the recipients immediately after BM transplantation showed significantly improved homing of HSPCs to the BM, which subsequently resulted in enhanced short- and long-term engraftment of HSPCs in mice. We found that TPO could downregulate the expression and secretion of matrix metalloproteinase 9 in BM cells. As a result, SDF-1α level was increased in the BM niche. Blocking the interaction of SDF-1α and CXCR4 on HSPCs by using AMD3100 could significantly reverse the TPO-enhanced HSPC homing effect. More importantly, a single dose of TPO remarkably promoted human HSPC homing and subsequent engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice. We then performed a clinical trial to evaluate the effect of TPO treatment in patients receiving haploidentical BM and mobilized peripheral blood transplantation. Surprisingly, single doses of TPO treatment to patients followed by hematopoietic stem cell transplantation significantly improved platelet engraftment in the cohort of patients with severe aplastic anemia (SAA). The mean volume of platelet and red blood cell transfusion was remarkably reduced in the cohort of patients with SAA or hematological malignancies receiving TPO treatment. Thus, our data provide a simple, feasible, and efficient approach to improve clinical outcomes in patients with allogenic hematopoietic stem cell transplantation. The clinical trial was registered in the Chinese Clinical Trial Registry website (www.chictr.org.cn) as ChiCTR-OIN-1701083., (© 2020 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34 + hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34 + CD38 - and CD34 + CD38 - CD90 + HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34 + hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34 + hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

Background: Several studies have suggested that caffeic acid phenethyl ester (CAPE) can induce the expression of hypoxia inducible factor-1α (HIF-1α) protein. We determined whether CAPE has a novel function in improving the homing and engraftment of haematopoietic stem/progenitor cells (HSPCs) by regulating HIF-1α gene expression in the bone marrow (BM) niche., Methods: For survival experiments, lethally irradiated C57BL/6 mice were injected with a low number of BM mononuclear cells (MNCs) and CAPE according to the indicated schedule. Homing efficiency analysis was conducted using flow cytometry and colony-forming unit (CFU) assays. The influence of intraperitoneal injection of CAPE on short-term and long-term engraftment of HSPCs was evaluated using competitive and non-competitive mouse transplantation models. To investigate the mechanism by which CAPE enhanced HSPC homing, we performed these experiments including Q-PCR, western blot, immunohistochemistry and CFU assays after in-vivo HIF-1α activity blockade., Results: CAPE injection significantly increased the survival rate of recipient mice after lethal irradiation and transplantation of a low number of BM MNCs. Using HSPC homing assays, we found that CAPE notably increased donor HSPC homing to recipient BM. The subsequent short-term and long-term engraftment of transplanted HSPCs was also improved by the optimal schedule of CAPE administration. Mechanistically, we found that CAPE upregulated the expression of HIF-1α, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor 1α (SDF-1α). The HIF-1α inhibitor PX-478 blocked CAPE-enhanced HSPC homing, which supported the idea that HIF-1α is a key target of CAPE., Conclusions: Our results showed that CAPE administration facilitated HSPC homing and engraftment, and this effect was primarily dependent on HIF-1α activation and upregulation of SDF-1α and VEGF-A expression in the BM niche.

Studies on coding genes, miRNAs, and lncRNAs during erythroid development have been performed in recent years. However, analysis focusing on the integration of the three RNA types has yet to be done. In the present study, we compared the dynamics of coding genes, miRNA, and lncRNA expression profiles. To explore dynamic changes in erythropoiesis and potential mechanisms that control these changes in the transcriptome level, we took advantage of high throughput sequencing technologies to obtain transcriptome data from cord blood hematopoietic stem cells and the following four erythroid differentiation stages, as well as from mature red blood cells. Results indicated that lncRNAs were promising cell marker candidates for erythroid differentiation. Clustering analysis classified the differentially expressed genes into four subtypes that corresponded to dynamic changes during stemness maintenance, mid-differentiation, and maturation. Integrated analysis revealed that noncoding RNAs potentially participated in controlling blood cell maturation, and especially associated with heme metabolism and responses to oxygen species and DNA damage. These regulatory interactions were displayed in a comprehensive network, thereby inferring correlations between RNAs and their associated functions. These data provided a substantial resource for the study of normal erythropoiesis, which will permit further investigation and understanding of erythroid development and acquired erythroid disorders.

Due to the low number of collectable stem cells from single umbilical cord blood (UCB) unit, their initial uses were limited to pediatric therapies. Clinical applications of UCB hematopoietic stem and progenitor cells (HSPCs) would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity. In recent years, numerous attempts have been made to expand human UCB HSPCs in vitro. In this study, we report that caffeic acid phenethyl ester (CAPE), a small molecule from honeybee extract, can promote in vitro expansion of HSPCs. Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells. Importantly, culture of CD34(+) HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units. Burst-forming unit-erythroid was the mostly affected colony type, which increased more than 3.7-fold in 1 μg mL(-1) CAPE treatment group when compared to the controls. CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α. Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into the blood circulation has been widely used for hematopoietic transplantation. However, the current methods of cytokine- or small-molecule-stimulated HSPC mobilization are far from satisfactory. New mobilizing agents are needed to increase the number of stem cells in peripheral blood for effective reconstitution of hematopoiesis. Here, we report that the molecule Me6TREN (Me6) can induce rapid mobilization of hematopoietic progenitor cells and that Me6 exhibits more significant effects than granulocyte colony-stimulating factor (G-CSF) or AMD3100. Me6 also mobilizes long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in primary and secondary transplant recipients. Mechanistically, Me6 inhibits both the SDF-1α-induced migration and VLA-4-mediated adhesion of mouse and human hematopoietic cells. Me6 appears to mobilize HSPCs by activating MMP-9 expression and disrupting the SDF-1α/CXCR4 axis. Therefore, Me6 may become a new potent and efficacious mobilizing agent of HSPCs.

Background: Currently, a constant shortage in the supply of platelets has become an important medical and society challenge, especially in developing country, and the in vitro production of megakaryocytic progenitor cells (MPs) from cord blood could represent an effective platelet substitute. In the present study, our objective was to determine the safety and feasibility of ex vivo generated MPs in patients., Methods and Findings: MPs were produced and characterized from cord blood mononuclear cells under a serum free medium with cytokines. We investigated the feasibility of expansion and infusion of cord blood-derived MPs in 24 patients with advanced hematological malignancies. The primary end point was the safety and tolerability of the infusion of cord blood-derived MPs. No adverse effects were observed in patients who received ex vivo-generated cells at concentrations of up to a median value of 5.45 × 10(6)cells/kg of body weight. With one year follow-up, acute and chronic GVHD had not been observed among patients who received MPs infusion, even without ABO blood group and HLA typing matching., Conclusions: These initial results in patients are very encouraging. They suggest that infusion of cord blood-derived MPs appears safe and feasible for treatment of thrombocytopenia.

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 10(9)-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive β -globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

Several studies have indicated that adult stem cells derived from bone marrow (BM) and cord blood (CB) can differentiate into hepatocyte-like cells. This ability is important for the treatment of hepatic diseases with BM or CB as a potential approach. However, methods are still being developed for the efficient induction of stem cell differentiation and expansion to get enough cells to be useful. In the present study, we enriched a subset of umbilical cord blood beta(2)m(-)c-Met(+) cells (UCBCCs) and investigated the combination effect of liver nonparenchymal cells (cirrhotic fat-storing cells [CFSCs]) and hepatocyte growth factor (HGF) on the induction of UCBCCs into hepatocyte-like cells. UCBCCs were cocultured with CFSC/HGF feeder layers either directly or separately using insert wells. Flow cytometric analysis showed that most UCBCCs were CD34(+/-)CD90(+/-)CD49f(+)CD29(+)Alb(+)AFP(+). After cocultured with transgenic feeder layers for 7 days, UCBCCs displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced UCBCCs expressed several hepatocyte specific genes including AFP, Alb, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells displayed liver specific functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin secretion. Hence, our data have demonstrated that UCBCCs might represent a novel subpopulation of CB-derived stem/progenitor cells capable of successful differentiation into hepatocyte-like cells when incubated with CFSC/HGF cells. In conclusion, not only HGF but also CFSCs and/or the secreted extracellular matrix (ECM) have been shown to be able to serve as essential microenvironment for hepatocyte differentiation.

Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. Over the last few years, transplantation of hematopoietic stem and progenitor cells from mobilized blood stem cells, umbilical cord blood and selected CD34+ cells has been used for treatment of patients with hematologic and non-hematologic malignances. The techniques have become available that allow the extensive proliferation, orderly differentiation, functional activation and gene transfer of hematopoietic stem/progenitor cells in ex vivo culture systems. These techniques have now developed to the point at which clinical trials are now underway in a variety of settings for the applications of hematopoietic stem cell transplantation, hematopoietic support after high-dose chemotherapy, immunotherapy of cancers, and gene therapy. The article will discuss the characteristics, detecting assays, surface markers, expansion, orderly differentiation, cell therapy and gene therapy of hematopoietic stem/progenitor cells. Except that, it will also discuss some advanced achievements of stem cell research, such as the multilineage differentiation of marrow-derived mesenchymal stem cells. Some results of different gene expression between stem cells and induced cells by gene chip analysis will be reported.

Background: Proper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation. Methods: We utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance. Results: In this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p. Conclusions: miR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs. [ABSTRACT FROM AUTHOR]

In vitro generation of red blood cells has the potential to circumvent shortfalls in the global demand for blood for transfusion applications. However, cell differentiation and proliferation are often regulated by precise changes in gene expression, but the underlying mechanisms and molecular changes remain unclear. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) can be used to evaluate multiple target genes. To make the results more reliable, suitable reference genes should be used to calibrate the error associated with qRT-PCR. In this study, we utilized bioinformatics to screen 3 novel candidate reference genes (calcium and integrin binding family member 2 [CIB2], olfactory receptor family 8 subfamily B member 8 [OR8B8], and zinc finger protein 425 [ZNF425]) along with eight traditional reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], β-actin [ACTB], 18S RNA, β2-microglobulin [β2-MG], peptidylprolyl isomerase A [PPIA], TATA box-binding protein [TBP], hydroxymethylbilane synthase [HMBS], and hypoxanthine phosphoribosyltransferase 1 [HPRT1]). Two software algorithms (geNorm and NormFinder) were used to evaluate the stability of expression of the 11 genes at different stages of erythrocyte development. Comprehensive analysis showed that expression of GAPDH and TBP was the most stable, whereas ZNF425 and OR8B8 were the least suitable candidate genes. These results suggest that appropriate reference genes should be selected before performing gene expression analysis during erythroid differentiation and that GAPDH and TBP are suitable reference genes for gene expression studies on erythropoiesis. [ABSTRACT FROM AUTHOR]

Today, liver transplantation (LT) is the only established treatment for end-stage liver diseases. The development of LT, including OLT, cadaveric LT, split LT, living donor LT (LDLT), brings hopes to patients with these diseases. However, increasing donor shortage, rejection and life-long immunosuppression with its side effects are the major limitations of this therapy strategy. Bone marrow-derived stem cells (BMDSC5) are capable of differentiating into hepatocyte-like cells and contribute to liver injury repair. The microenvironment of liver injury caused by rejection, ischemia/reperfusion, loss of liver mass, recurrence of HCV and ‘small-for-size syndrome’ after LT can attract a variety of bone marrow-derived stem cell population to the peripheral circulation and then migration to the injury liver to promote the hepatic function restoration. Additionally, BMDSCs can also take part in the functional regeneration of living donor liver after LDLT. This participation in liver regeneration may be associated to the interaction between SDF-land its receptor CXCR4, involving HGF, IL-8, MMP9, and VEGFNEGFR-2. BMDSC with its bio-characteristics could maintain the allograft tolerance from different angles and in different ways. In conclusion, BMDSC5 transplantation, as a new assistant therapeutic method for LT, will expand the space of LT, and provide more survival opportunities for the patients suffering liver diseases in the future. [ABSTRACT FROM AUTHOR]

There is an urgent medical need to develop effective therapies that can ameliorate damage to the radiation-exposed hematopoietic system. Nanozymes with robust antioxidant properties have a therapeutic potential for mitigating radiation-induced hematopoietic injury. However, enhancing nanozyme recruitment to injured tissues in vivo while maintaining their catalytic activity remains a great challenge. Herein, we present the design and preparation of a biomimetic nanoparticle, a mesenchymal stem cell membrane camouflaged Prussian blue nanozyme (PB@MSCM), which exhibits biocompatible surface properties and demonstrates enhanced injury site-targeting towards the irradiated murine bone marrow niche. Notably, the constructed PB@MSCM possessed redox enzyme-mimic catalytic activity and could scavenge overproduced reactive oxygen species in the irradiated bone marrow cells, both in vitro and ex vivo. More importantly, the administration of PB@MSCM significantly mitigated hematopoietic cell apoptosis and accelerated the regeneration of hematopoietic stem and progenitor cells. Our findings provide a new targeted strategy to improve nanozyme therapy in vivo and mitigate radiation-induced hematopoietic injury. [ABSTRACT FROM AUTHOR]