Search

Additional file 1. Supplemental methods, figures and tables.

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 Δ17(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.

Background: Acute gastrointestinal syndrome (AGS) is one of the most severe clinical manifestations after exposure to high doses of radiation, and is life-threatening in radiological emergency scenarios. However, an unmet challenge is lacking of an FDA-approved drug that can ameliorate the damage of radiation-exposed intestinal tissues and accelerate the regeneration of injured epithelia. In this study, we investigated whether the small molecule Me6TREN (Me6) can regulate intestinal stem cell (ISC) proliferation and promote crypt regeneration after irradiation. Methods: Lethally irradiated mice were administered with Me6 or PBS to study the survival rate, and sections of their small intestine were subjected to immunostaining to evaluate epithelial regeneration. An intestinal organoid culture system was employed to detect the role of Me6 in organoid growth and ISC proliferation. We further investigated the key signaling pathways associated with Me6 using microarray, western blotting, and RNA interference techniques. Results: We identified the small molecule Me6 as a potent intestinal radiation countermeasure. Systemic administration of Me6 significantly improved ISC and crypt cell regeneration and enhanced the survival of mice after high doses of radiation. Using an in vitro intestinal organoid culture system, we found that Me6 not only induced ISC proliferation but also increased the budding rate of intestinal organoids under unirradiated and irradiated conditions. Me6 remarkably activated the expression of ISC-associated and proliferation-promoting genes, such as Ascl2, Lgr5, Myc, and CyclinD1. Mechanistically, Me6 strongly stimulated the phosphorylation of β-catenin at the S552 site and increased the transcriptional activity of β-catenin, a key signaling pathway for ISC self-renewal and proliferation. This is further evidenced by the fact that knockdown of β-catenin abolished the effect of Me6 on intestinal organoid growth in vitro and crypt regeneration in irradiated mice. Conclusion: The small molecule Me6TREN induced ISC proliferation, enhanced intestinal organoid growth in vitro, and promoted intestinal tissue regeneration after radiation injury by activating β-catenin signaling.

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 (http://www.chictr.org.cn) as ChiCTR‐OIN‐1701083., We reported a novel function of recombinant human thrombopoietin (TPO) in increasing hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM), which subsequently resulted in enhanced long‐term engraftment of HSPCs in mice. Notably, single doses of TPO treatment to patients followed by HSCT improved clinical outcomes, especially in patients with severe aplastic anemia.

Red blood cells (RBCs) and platelets derived from stem cells are possible solutions to the increasing demand for blood transfusion. Based on the availability of stem cells, their relatively defined differentiation mechanisms, and the massive exploration of induction systems, the generation of RBCs or platelets in vitro from cord blood hematopoietic stem/progenitor cells (CB‐HSPCs) has potential for clinical applications. However, information on the clinical translation of stem cell‐derived RBCs and platelets in the literature and at the ClinicalTrials.gov website is very limited. The only clinical trial on cultured RBCs, which aimed to assess the lifespan of RBCs cultured in vivo, was reported by Luc Douay and colleagues. Of note, the cultured RBCs they used were derived from autologous peripheral blood HSPCs, and no cultured platelets have been applied clinically to date. However, CB‐HSPC‐derived megakaryocytes, platelet precursors, have been used in the treatment of thrombocytopenia. A successful phase I trial was reported, followed by phase II and III clinical trials conducted in China. In this review, the gap between the many basic studies and limited clinical trials on stem cell‐derived RBCs and platelets is summarized. The possible reasons and solutions for this gap are discussed. Further technological improvements for blood cell expansion and maturation ex vivo and the establishment of biological standards for stem cell derivatives might help to facilitate the therapeutic applications of cultured RBCs and platelets derived from CB‐HSPCs in the near future., We review the progress in producing red blood cells (RBCs) and platelets from human cord blood hematopoietic stem/progenitor cells (HSPCs) and relatively limited reports on the clinical trials with cultured RBCs and megakaryocytic progenitors (MPs). Technical improvement together with the establishment of biological standards for stem cell derivations might help to achieve therapeutic applications of cultured RBCs and platelets. EPCs, erythroid progenitor cells.

BackgroundEx vivo production of induced megakaryocytes (MKs) and platelets from stem cells is an alternative approach for supplying transfusible platelets. However, it is difficult to generate large numbers of MKs and platelets from hematopoietic stem cells and progenitor cells (HSPCs).MethodsTo optimize the differentiation efficiency of megakaryocytic cells from HSPCs, we first employed a platelet factor 4 (PF4)-promoter reporter and high-throughput screening strategy to screen for small molecules. We also investigated the effects and possible mechanisms of candidate small molecules on megakaryocytic differentiation of human HSPCs.ResultsThe small molecule Ricolinostat remarkably promoted the expression of PF4-promoter reporter in the megakaryocytic cell line. Notably, Ricolinostat significantly enhanced the cell fate commitment of MK progenitors (MkPs) from cord blood HSPCs and promoted the proliferation of MkPs based on cell surface marker detection, colony-forming unit-MK assay, and quantitative real-time PCR analyses. MkPs generated from Ricolinostat-induced HSPCs differentiated into mature MKs and platelets. Mechanistically, we found that Ricolinostat enhanced MkP fate mainly by inhibiting the secretion of IL-8 and decreasing the expression of the IL-8 receptor CXCR2.ConclusionThe addition of Ricolinostat to the culture medium promoted MkP differentiation from HSPCs and enhanced the proliferation of MkPs mainly by suppressing the IL-8/CXCR2 pathway. Our results can help the development of manufacturing protocols for the efficient generation of MKs and platelets from stem cells in vitro.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0708-x) contains supplementary material, which is available to authorized users.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9L(LUC) glioma cells syngenic to Fischer 344 (F344) rats. Bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9L(LUC)/F344 rats, and tumor regression was found in some 9L(LUC)/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9L(LUC)/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9L(LUC)/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.

MicroRNAs (miRNAs) are small, noncoding RNAs which can often act as an oncogene or a tumor suppressor. Several miRNAs are associated with the development of hepatocellular carcinoma (HCC). We demonstrated that miR-125b significantly suppresses HCC cell proliferation and promotes apoptosis by inhibiting the gene expression of the anti-apoptotic protein, Bcl-2. Bioinformatic analysis indicated that the 3'UTR of Bcl-2 has binding sites for miR-125b. Luciferase reporter assay confirmed the ability of miR-125b to dramatically suppress Bcl-2 transcription, suggesting that Bcl-2 is a target gene for miR-125b. We concluded that miR-125b acts as a tumor suppressor in hepatic tumor development by targeting Bcl-2 and inducing cancer cell apoptosis.

Dibutyl phthalate (DBP) showed preferential suppression of the growth of leukemic cells and could be used as a purging agent for autologous bone marrow transplantation in the treatment of leukemia. With in vitro exposure of leukemic cells to DBP, most characteristic changes in morphology of apoptosis could be observed by electron microscopy while DNA degraded in this fashion gave a 'ladder' pattern on DNA gel electrophoresis. The obtained results demonstrated that the purging effect of DBP on leukemic cells is mediated, at least in part, by the induction of apoptosis.

Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment.

It has been proved that di-N-butyl phthalate (DBP) is singular in killing leukemic cells selectively or accelerating the deterioration of residual leukemic cells in long-term marrow culture in vitro. Based on this principle, the DBP-purged autologous bone marrow transplant has been applied to the treatment of a group of 14 patients suffering from acute non-lymphocytic leukemia. After 5–10 days of in vitro co-culture of marrow cells with DBP at a concentration of 50 μg/ml, the recovery of total nucleated cells and the amount of CFU-GM were 67.5% and 68.1%, respectively. In all patients, the reconstitution of hematopoiesis was observed after pre-conditioning and transfusion of purged marrow cells. Among these, two patients had a relapse, two patients died from complications of transplant, one patient died from non-leukemic disease, and the others are all alive and free of disease; the mean survival time as calculated recently was 15 months. These preliminary clinical data support that marrow culture in the presence of DBP is a safe and effective measure for treating leukemia in purged autologous bone marrow transplant.

DBP possesses a new pharmacological activity leading to selective deterioration of leukemic cells, with less harmful effect on the growth of normal hemopoietic progenitors.