A small molecule compound IX inhibits telomere and attenuates oncogenesis of drug-resistant leukemia cells.

Drug resistance is a common obstacle in leukemia treatment and failing to eradicate leukemia stem cells is the main cause of leukemia relapse. Previous studies have demonstrated that telomerase activity is associated with deregulated self-renewal of leukemia stem cells (LSCs). Here, we identified a novel compound IX, an imatinib derivative with a replacement fragment of a telomerase inhibitor, which can effectively eradicate LSCs but had no influence on normal hematopoietic stem cells (HSCs) survival. We showed that compound IX can decrease the viability of drug-resistant K562/G cells and blast crisis CML primary patient cells. Besides, IX can affect LSC survival, inhibit the colony-forming ability, and reduce LSC frequency. In vivo results showed that IX can relieve the tumor burden in patient-derived xenograft (PDX) model and prolong the lifespan. We observed that compound IX can not only decrease telomerase activity, but also affect the alternative lengthening of telomeres. In addition, IX can inhibit both the canonical and non-canonical Wnt pathways. Our data suggested this novel compound IX as a promising candidate for drug-resistant leukemia therapy.

TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway.

The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB-derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)-derived HSC. TNFSF15-treated UCB-HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi-SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle-treated group. Additionally, signal proteins of the Notch pathway are highly up-regulated in TNFSF15-treated UCB-HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB-HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB-HSC application or study.

Antioxidant N-acetyl-L-cysteine increases engraftment of human hematopoietic stem cells in immune-deficient mice.

Immunocompromised mice, such as the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, have been widely used to examine the self-renewal and differentiation potential of human hematopoietic stem cells (HSCs) in vivo. However, the efficiency of human HSC engraftment remains very low. Here, we report that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in their bone marrow (BM) than other commonly used mouse strains (C57BL/6 and BALB/C). Treatment with the antioxidant N-acetyl-l-cysteine (NAC) decreased ROS levels in the BM of NOD/SCID mice. Furthermore, the NAC-treated mice displayed a significant increase in human HSC engraftment and multilineage hematopoietic differentiation in the mice. In comparison with the control mice, NAC-treated recipients displayed a 10.8-fold increase in hematopoietic engraftment in the injected tibiae. A beneficial effect of NAC for human hematopoietic engraftment was also observed in an additional immunodeficient mouse strain, namely NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NOD/SCID/γc(-/-) or NSG). Thus, this study uncovers a previously unappreciated negative effect of ROS on human stem cell engraftment in immunodeficient mice.

StemCellCKB: An Integrated Stem Cell-Specific Chemogenomics KnowledgeBase for Target Identification and Systems-Pharmacology Research.

Given the capacity of self-renewal and multilineage differentiation, stem cells are promising sources for use in regenerative medicines as well as in the clinical treatment of certain hematological malignancies and degenerative diseases. Complex networks of cellular signaling pathways largely determine stem cell fate and function. Small molecules that modulate these pathways can provide important biological and pharmacological insights. However, it is still challenging to identify the specific protein targets of these compounds, to explore the changes in stem cell phenotypes induced by compound treatment and to ascertain compound mechanisms of action. To facilitate stem cell related small molecule study and provide a better understanding of the associated signaling pathways, we have constructed a comprehensive domain-specific chemogenomics resource, called StemCellCKB ( http://www.cbligand.org/StemCellCKB/ ). This new cloud-computing platform describes the chemical molecules, genes, proteins, and signaling pathways implicated in stem cell regulation. StemCellCKB is also implemented with web applications designed specifically to aid in the identification of stem cell relevant protein targets, including TargetHunter, a machine-learning algorithm for predicting small molecule targets based on molecular fingerprints, and HTDocking, a high-throughput docking module for target prediction and systems-pharmacology analyses. We have systematically tested StemCellCKB to verify data integrity. Target-prediction accuracy has also been validated against the reported known target/compound associations. This proof-of-concept example demonstrates that StemCellCKB can (1) accurately predict the macromolecular targets of existing stem cell modulators and (2) identify novel small molecules capable of probing stem cell signaling mechanisms, for use in systems-pharmacology studies. StemCellCKB facilitates the exploration and exchange of stem cell chemogenomics data among members of the broader research community.

No evidence for neo-oogenesis may link to ovarian senescence in adult monkey.

Female germline or oogonial stem cells transiently residing in fetal ovaries are analogous to the spermatogonial stem cells or germline stem cells (GSCs) in adult testes where GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have not been consistently reproducible. Monkeys provide the most relevant model of human ovarian biology. We searched for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey testis, which are characterized by cytoplasmic staining for the germ cell marker DAZL and nuclear expression of the proliferative markers PCNA and KI67, and pluripotency-associated genes LIN28 and SOX2, and lack of nuclear LAMIN A, a marker for cell differentiation. Early meiocytes undergo homologous pairing at prophase I distinguished by synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we found proliferative nongermline somatic stem cells that do not express LAMIN A and germ cell markers in the adult ovaries, notably in the cortex and granulosa cells of growing follicles. These data support the paradigm that adult ovaries do not undergo germ cell renewal, which may contribute significantly to ovarian senescence that occurs with age.

MCL-1 Inhibitor S63845 Distinctively Affects Intramedullary and Extramedullary Hematopoiesis.

Conventional chemotherapy for killing cancer cells using cytotoxic drugs suffers from low selectivity, significant toxicity, and a narrow therapeutic index. Hyper-specific targeted drugs achieve precise destruction of tumors by inhibiting molecular pathways that are critical to tumor growth. Myeloid cell leukemia 1 (MCL-1), an important pro-survival protein in the BCL-2 family, is a promising antitumor target. In this study, we chose to investigate the effects of S63845, a small-molecule inhibitor that targets MCL-1, on the normal hematopoietic system. A mouse model of hematopoietic injury was constructed, and the effects of the inhibitor on the hematopoietic system of mice were evaluated via routine blood tests and flow cytometry. The results showed that S63845 affected the hematopoiesis of various lineages in the early stage of action, causing extramedullary compensatory hematopoiesis in the myeloid and megakaryocytic lineages. The maturation of the erythroid lineage in the intramedullary and extramedullary segments was blocked to varying degrees, and both the intramedullary and extramedullary lymphoid lineages were inhibited. This study provides a complete description of the effects of MCL-1 inhibitor on the intramedullary and extramedullary hematopoietic lineages, which is important for the selection of combinations of antitumor drugs and the prevention of adverse hematopoiesis-related effects.

Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche.

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34+CD38-CD45RA-CD90+CD49f lowCD62L-CD133+ subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

Rapid and non-invasive discrimination of acute leukemia bone marrow supernatants by Raman spectroscopy and multivariate statistical analysis.

A simple and non-invasive detection method for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) was established by systematically investigating the characteristics of bone marrow supernatants from 61 AML patients, 22 ALL patients, and 5 volunteers without hematological tumors by Raman spectroscopy and orthogonal partial least squares discriminant analysis (OPLS-DA). The control group could be well distinguished from the AML and ALL groups by Raman peaks of 859, 1031, 1437, 1443, 1446, 1579, and 1603 cm-1 and from the AML subtypes groups (AML-M2, AML-M3, AML-M4, and AML-M5) by the Raman peaks of 859, 1221, 1230, 1437, 1443, and 1603 cm-1, indicating high sensitivity and specificity of the method. Potentially important variables of acute leukemia (AL) prognosis, such as cholesterol, high-density lipoprotein, low-density lipoprotein, adenosine deaminase, and hemoglobin, could be effectively identified by Raman peaks of 1437, 1443, and 1579 cm-1. Therefore, Raman spectroscopy can be considered as a new non-invasive clinical tool for the detection of different types of AL and can be used to correlate biochemical parameters of AL patients with the classification and prognosis of AL.

Deciphering the Heterogeneity of Mitochondrial Functions During Hematopoietic Lineage Differentiation.

BACKGROUND: The heterogeneity of mitochondrial function is an important feature of hematopoietic cell lineage differentiation, but its stage wise contribution is not adequately studied. To establish a model to compare the lineage differentiation of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, the mitochondrial mass (MM), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitophagy level were analyzed. RESULTS AND DISCUSSION: HSCs had lower mitochondrial metabolic activity than committed progenitor populations, indicated by lower MM, MMP, and ROS and higher mitophagy. HPC1s shared more stem cell characteristics than HPC2s and committed progenitor populations in terms of mitochondrial number and function. The mitochondrial metabolism of mature blood cells had greater heterogeneity than hematopoietic stem and progenitor cells, with granulocytes being similar to monocytes. Moreover, HSCs exhibited heterogeneity in the selection of mitophagy-related PINK1/PARK2, BNIP3/NIX, and FUNDC1 pathways. Myeloid differentiation had greater morphological and functional heterogeneity of hematopoietic cells than lymphoid differentiation. Additionally, leukemia stem cells had higher aerobic metabolism and better stem cell function through elevated mitophagy than normal hematopoietic cells. ROS and MMP levels in differentiated leukemia cells were higher, but the level of mitophagy was lower than in differentiated hematopoietic cells. CONCLUSION: This study provides a complete set of methods and basic reference values for the systematic study of the mitochondrial metabolic function of different types of hematopoietic cells under physiological and pathological conditions. The findings contribute to the future research of tumor and aging based on mitochondrial metabolism.

Elucidating the mitochondrial function of murine lymphocyte subsets and the heterogeneity of the mitophagy pathway inherited from hematopoietic stem cells.

Background: Mitochondria are mainly involved in ATP production to meet the energy demands of cells. Researchers are increasingly recognizing the important role of mitochondria in the differentiation and activation of hematopoietic cells, but research on how mitochondrial metabolism influence different subsets of lymphocyte at different stages of differentiation and activation are yet to be carried out. In this work, the mitochondrial functions of lymphocytes were compared at different differentiation and activation stages and included CD8+ T lymphocytes, CD4+ T lymphocytes, B lymphocytes, NK cells as well as their subsets. For this purpose, a complete set of methods was used to comprehensively analyze mitophagy levels, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the mitochondrial mass (MM) of subsets of lymphocytes. It is expected that this will provide a complete set of standards, and drawing the mitochondrial metabolic map of lymphocyte subsets at different stages of differentiation and activation. Results and discussion: Of all lymphocytes, B cells had a relatively high mitochondrial metabolic activity which was evident from the higher levels of mitophagy, ROS, MMP and MM, and this reflected the highly heterogeneous nature of the mitochondrial metabolism in lymphocytes. Among the B cell subsets, pro-B cells had relatively higher levels of MM and MMP, while the mitochondrial metabolism level of mature B cells was relatively low. Similarly, among the subsets of CD4+ T cell, a relatively higher level of mitochondrial metabolism was noted for naive CD4+ T cells. Finally, from the CD8+ T cell subsets, CD8+ Tcm had relatively high levels of MM and MMP but relatively low ones for mitophagy, with effector T cells displaying the opposite characteristics. Meanwhile, the autophagy-related genes of lymphoid hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells and lymphocyte subsets were analyzed, which preliminarily showed that these cells were heterogeneous in the selection of mitophagy related Pink1/Park2, BNIP3/NIX and FUNDC1 pathways. The results showed that compared with CD4+ T, CD8+ T and NK cells, B cells were more similar to long-term hematopoietic stem cell (LT-HSC) and short-term hematopoietic stem cell (ST-HSC) in terms of their participation in the Pink1/Park2 pathway, as well as the degree to which the characteristics of autophagy pathway were inherited from HSC. Compared with CLP and B cells, HSC are less involved in BNIP3/NIX pathway. Among the B cell subsets, pro-B cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with pre-B, immature B and immature B cells. Among CD4+ T cell subsets, nTreg cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with naive CD4+ T and memory CD4+ T cells. Among the CD8+ T cell subsets, compared with CLP and effector CD8+ T cells, CD8+ Tcm inherit the least characteristics of HSC in participating in Pink1/Park2 pathway. Meanwhile, CLP, naive CD4+ T and effector CD8+ T were more involved in BNIP3/NIX pathway than other lymphoid hematopoietic cells. Conclusion: This study is expected to provide a complete set of methods and basic reference values for future studies on the mitochondrial functions of lymphocyte subsets at different stages of differentiation and activation in physiological state, and also provides a standard and reference for the study of infection and immunity based on mitochondrial metabolism.

Levistilide A Promotes Expansion of Human Umbilical Cord Blood Hematopoietic Stem Cells by Enhancing Antioxidant Activity.

Several approaches to expand human hematopoietic stem cells (hHSCs) clinically along with retainable capability of multipotential differentiation have been reported, but only a few have advanced to evaluation in clinical trials, which limits the application of HSC-based therapy. Here we show a phthalide derivative, Levistilide A (LA), can serve as a promising molecule to expand functional human umbilical cord blood (UCB) HSCs ex vivo. An in-house screen identified LA out of nine natural products as an outstanding candidate for hHSCs expansion. Additionally, our data indicated that LA treatment not only increased the numbers of phenotype-defined HSCs, but also enhanced their colony formation ability. Xenotransplantation assays showed that LA treatment could maintain unaffected engraftment of hHSCs with multilineage differentiation capacity. Further experiments revealed that LA enhanced the antioxidant activity of hHSCs by reducing intracellular and mitochondrial reactive oxygen species (ROS) levels. The identification of LA provides a new strategy in solving the clinical issue of limited numbers of UCB HSCs.

KDM4 inhibitor SD49-7 attenuates leukemia stem cell via KDM4A/MDM2/p21CIP1 axis.

Rationale: Traditional treatments for leukemia fail to address stem cell drug resistance characterized by epigenetic mediators such as histone lysine-specific demethylase 4 (KDM4). The KDM4 family, which acts as epigenetic regulators inducing histone demethylation during the development and progression of leukemia, lacks specific molecular inhibitors. Methods: The KDM4 inhibitor, SD49-7, was synthesized and purified based on acyl hydrazone Schiff base. The interaction between SD49-7 and KDM4s was monitored in vitro by surface plasma resonance (SPR). In vitro and in vivo biological function experiments were performed to analyze apoptosis, colony-formation, proliferation, differentiation, and cell cycle in cell sub-lines and mice. Molecular mechanisms were demonstrated by RNA-seq, ChIP-seq, RT-qPCR and Western blotting. Results: We found significantly high KDM4A expression levels in several human leukemia subtypes. The knockdown of KDM4s inhibited leukemogenesis in the MLL-AF9 leukemia mouse model but did not affect the survival of normal human hematopoietic cells. We identified SD49-7 as a selective KDM4 inhibitor that impaired the progression of leukemia stem cells (LSCs) in vitro. SD49-7 suppressed leukemia development in the mouse model and patient-derived xenograft model of leukemia. Depletion of KDM4s activated the apoptosis signaling pathway by suppressing MDM2 expression via modulating H3K9me3 levels on the MDM2 promoter region. Conclusion: Our study demonstrates a unique KDM4 inhibitor for LSCs to overcome the resistance to traditional treatment and offers KDM4 inhibition as a promising strategy for resistant leukemia therapy.

Requirements for human haematopoietic stem/progenitor cells.

'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.

A mitophagy inhibitor targeting p62 attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

There has been an increasing focus on the tumorigenic potential of leukemia initiating cells (LICs) in acute myeloid leukemia (AML). Despite the important role of selective autophagy in the life-long maintenance of hematopoietic stem cells (HSCs), cancer progression, and chemoresistance, the relationship between LICs and selective autophagy remains to be fully elucidated. Sequestosome 1 (SQSTM1), also known as p62, is a selective autophagy receptor for the degradation of ubiquitinated substrates, and its loss impairs leukemia progression in AML mouse models. In this study, we evaluated the underlying mechanisms of mitophagy in the survival of LICs with XRK3F2, a p62-ZZ inhibitor. We demonstrated that XRK3F2 selectively impaired LICs but spared normal HSCs in both mouse and patient-derived tumor xenograft (PDX) AML models. Mechanistically, we observed that XRK3F2 blocked mitophagy by inhibiting the binding of p62 with defective mitochondria. Our study not only evaluated the effectiveness and safety of XRK3F2 in LICs, but also demonstrated that mitophagy plays an indispensable role in the survival of LICs during AML development and progression, which can be impaired by blocking p62.

Bone marrow mesenchymal cells: polymorphism associated with transformation of rough endoplasmic reticulum.

To understand the behavior and function of bone-marrow mesenchymal cells (BMMCs), we overviewed the morphological presentation of BMMCs in bone-marrow granules (b-BMMCs), isolated BMMCs (i-BMMCs), and BMMCs (c-BMMCs) cultured in H4434 methylcellulose semisolid and MEM media. All samples were derived from bone-marrow aspirates of 30 patients with hematocytopenia. Light microscopy exhibited b-BMMCs and i-BMMCs characterized by abundant cytoplasm and irregular shape in bone-marrow smears, as well as c-BMMCs in culture conditions. Scanning electron microscopy demonstrated cultured c-BMMCs with a sheet-like feature enveloping hematopoietic cells. Transmission electron microscopy revealed b-BMMCs constructing a honeycomb-like structure by thin bifurcate processes among hematopoietic cells. Furthermore, i-BMMCs had bifurcate parapodiums on the surface and prominent rough endoplasmic reticulum (rER) connected with the plasmalemma of the parapodiums. The detailed images suggested that rER may serve as a membrane resource for plasmalemmal expansion in BMMCs in bone marrow.

[The Effect and Mechanism of Novel Telomerase Inhibitor Nilo 22 on Leukemia Cells].

OBJECTIVE: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. METHODS: The cytotoxic effects of 28 Nilotinib derivatives on K562, KA, KG, HA and 32D cell lines were detected by MTT assays, and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP+ cells was tested by colony-forming units assays (CFU). The cytotoxic effect was further detected by transplant assays ex vivo. Telomerase activity assay, C-circle assay were used to measure the effects of compound on the length mechanism of telomere, RT-PCR was used to detected the changes of telomere. RESULTS: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives, which impairs leukemia cell lines, but spares normal hematopoietic cell line. Comparing with Nilotinib, Nilo 22 could induce the apoptosis of GFP+ cells significantly, slightly arrests the cell cycle at G0/G1 phase, and significantly inhibits colony formation and prolong the progression in MLL-AF9 leukemia mice model. The expression showed that the compound could slow the disease progression in MLL-AF9 leukemia mice significantly. Mechanistically, Nilo 22 could reduce the length of telomere by inhibiting telomerase activity and alternative lengthening of telomere (ALT). CONCLUSION: Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells, especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.

Enhanced self-renewal of human long-term hematopoietic stem cells by a sulfamoyl benzoate derivative targeting p18INK4C.

The use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18INK4C (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.

Antioxidant Small Molecule Compound Chrysin Promotes the Self-Renewal of Hematopoietic Stem Cells.

There is an increasing demand for the expansion of functional human hematopoietic stem cells (hHSCs) for various clinical applications. Based on our primary screening of antioxidant small molecule compounds library, a small molecule compound C2968 (chrysin) was identificated to expand cord blood CD34+ cells in vitro. Then we further verified the optimum concentration and explored its effect on hHSCs phenotype and biological function. C2968 could significantly increase the proportion and absolute number of CD34+CD38-CD49f+ and CD34+CD38-CD45RA-CD90+ cells under 2.5 µM. Furthermore, the total number of colony-forming units and the frequency of LT-HSCs in C2968-treated group were significantly higher than control, indicating the multipotency and long-term activity of hematopoietic stem and progenitor cells were sustained. Additionally, C2968 treatment could maintain transplantable HSCs that preserve balanced multilineage potential and promote rapid engraftment after transplantation in immunodeficient (NOG) mice. Mechanistically, the activity of chrysin might be mediated through multiple mechanisms namely delaying HSC differentiation, inhibiting ROS-activated apoptosis, and modulating of cyclin-dependent kinase inhibitors. Overall, chrysin showed good ex vivo expansion effect on hHSCs, which could maintain the self-renewal and multilineage differentiation potential of hHSCs. Through further research on its antioxidant mechanism, it may become a promising tool for further fundamental research and clinical umbilical cord blood transplantation of hHSCs.

Coinhibition of activated p38 MAPKα and mTORC1 potentiates stemness maintenance of HSCs from SR1-expanded human cord blood CD34+ cells via inhibition of senescence.

The stemness of ex vivo expanded hematopoietic stem cells (HSCs) is usually compromised by current methods. To explore the failure mechanism of stemness maintenance of human HSCs, which were expanded from human umbilical cord blood (hUCB) CD34+ cells, by differentiation inhibitor Stem Regenin 1 (SR1), an antagonist of aryl hydrocarbon receptor, we investigated the activity of p38 mitogen-activated protein kinase α (p38 MAPKα, p38α) and mammalian target of rapamycin complex 1 (mTORC1), and their effect on SR1-expanded hUCB CD34+ cells. Our results showed that cellular senescence occurred in the SR1-expanded hUCB CD34+ cells in which p38α and mTORC1 were successively activated. Furthermore, their coinhibition resulted in a further decrease in hUCB CD34+ cell senescence without an effect on apoptosis, promoted the maintenance of expanded phenotypic HSCs without differentiation inhibition, increased the hematopoietic reconstitution ability of multiple lineages, and potentiated the long-term self-renewal capability of HSCs from SR1-expanded hUCB CD34+ cells in NOD/Shi-scid/IL-2Rγnull mice. Our mechanistic study revealed that senescence inhibition by our strategy was mainly attributed to downregulation of the splicesome, proteasome formation, and pyrimidine metabolism signaling pathways. These results suggest that coinhibition of activated p38α and mTORC1 potentiates stemness maintenance of HSCs from SR1-expanded hUCB CD34+ cells via senescence inhibition. Thus, we established a new strategy to maintain the stemness of ex vivo differentiation inhibitor-expanded human HSCs via coinhibition of multiple independent senescence initiating signal pathways. This senescence inhibition-induced stemness maintenance of ex vivo expanded HSCs could also have an important role in other HSC expansion systems.

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli.

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37 degrees C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.