Stem Cell Surface Research Articles

Parathyroid Hormone-Related Protein (PTHrP) Accelerates Soluble RANKL Signals for Downregulation of Osteogenesis of Bone Mesenchymal Stem Cells.

A recent study reported the expression of receptor activator of nuclear factor-κB (RANK) in mesenchymal stem cells (MSCs) surface that negatively regulates osteogenesis of MSCs. Empirical evidence from the previous study confirmed the role of parathyroid hormone-related protein (PTHrP) in osteoblastogenesis. However, it is necessary to understand the paracrine role of PTHrP and RANKL for osteogenesis in order to explore the hidden secrets in bone biology. Considering the above concept, paracrine cues of soluble-receptor activator of nuclear factor-κB ligand (sRANKL) and PTHrP in osteogenic differentiation of MSCs were investigated. Our results confirmed that sRANKL increased the expression of surface-RANK in MSCs at the earlier stage of osteogenesis, which was downregulated later in differentiated MSCs. In contrast, RANKL expression was low at the earlier stage of MSCs proliferation and high at the differentiation stage of MSCs, which may play a fundamental role in osteoclast formation. sRANKL downregulated osteogenesis of MSCs by decreasing progressive ankylosis (ANK) protein expression while PTHrP upregulated the osteogenic exploitive effect of sRANKL. Interestingly, when they were co-cultured with MSCs, T-lymphocytes expressed high membrane-RANKL levels that contribute to osteogenesis inhibition during MSC differentiation. Thus, our results disclose that sRANKL treatment downregulates osteogenesis of MSCs by increasing RANK expression at the earlier stage of differentiation and by inhibiting ANK. Further, we demonstrated that PTHrP accelerated the downregulating osteogenic effect of sRANKL.

Cluster of Differentiation 96 in Children with Acute Leukemia: A Single Center Cohort Study.

Cluster of differentiation 96 (CD96) is an important leukemic stem cells (LSCs) surface marker. We evaluated CD96 expression in children with acute leukemia (AL) and described its relation with treatment response. We conducted a prospective cohort study in Mansoura University Children's Hospital, Egyptduring the period from 2014 to 2016. We studied 96 children with AL and 96 controls at clinical, laboratory and radiological levels. We assessed CD96% in LSCs using flow cytometry. AL group included 59 acute lymphoblastic leukemia (ALL) and 37 acute myeloid leukemia (AML) patients. ALL subgroup involved 44 B-ALL and 15 T-ALL patients while AML subgroup included 17 M2, 12 M4 and 8 M5 patients. CD96% was higher in AL group [57.63 (21.18-89.93)] than control [34.12 (16.15-39.51)] (P < 0.001). CD96% was higher in AML [68.25 (31.1-89.86)] than ALL [54.18 (21.18-89.93] (P < 0.001). CD96% in AML was M4 > M2 > M5 (P = 0.04) while within ALL subgroup, no significant difference was found between B-ALL and T-ALL (P = 0.807). CD96% in patients with non-complete remission was higher than those with complete remission (P = 0.004). CD96 is a reliable diagnostic marker for AL mainly AML and could be used as a prognostic marker for treatment response.

Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures

Through the paracrine effects of stem cells, including the secretion of neurotrophic, immunomodulatory, and anti-apoptotic factors, cell-based therapies offer a new all-encompassing approach to treatment of neurodegenerative diseases. In this study, we used physically separated co-cultures of porcine neuroretina (NR) and human mesenchymal stem cells (MSC) to evaluate the MSC paracrine neuroprotective effects on NR degeneration. NR explants were obtained from porcine eyes and cultured alone or co-cultured with commercially available MSCs from Valladolid (MSCV; Citospin S.L.; Valladolid, Spain), currently used for several approved treatments. Cultures were maintained for 72 h. MSC surface markers were evaluated before and after co-culture with NRs. Culture supernatants were collected and the concentration of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) were determined by enzyme-linked immunosorbent assays. NR sections were stained by haematoxylin/eosin or immunostained for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), glial fibrillary acidic protein, β-tubulin III, and neuronal nuclei marker. NR morphology, morphometry, nuclei count, apoptosis rate, retinal ganglion cells, and glial cell activation were evaluated. Treatment effects were statistically analysed by parametric or non-parametric tests. The MSCs retained stem cell surface markers after co-culture with NR. BDNF and CNTF concentrations in NR-MSCV co-cultures were higher than other experimental conditions at 72 h (p < 0.05), but no GDNF was detected. NR general morphology, total thickness, and cell counts were broadly preserved in co-cultures, and the apoptosis rate determined by TUNEL assay was lower than for NR monocultures (all p < 0.05). Co-cultures with MSCV also protected retinal ganglion cells from degenerative changes and reduced reactive gliosis (both p < 0.05). In this in vitro model of spontaneous NR degeneration, the presence of co-cultured MSCs retarded neuroglial degeneration. This effect was associated with elevated concentrations of the neurotrophic factors BDNF and CNTF. Our data suggest that the paracrine secretion of these, and possibly other molecules, are a potential resource for the treatment of several neuroretinal diseases.

Examination of CD302 as a potential therapeutic target for acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with ~20,000 new cases yearly. The disease develops in people of all ages, but is more prominent in the elderly, who due to limited treatment options, have poor overall survival rates. Monoclonal antibodies (mAb) targeting specific cell surface molecules have proven to be safe and effective in different haematological malignancies. However, AML target molecules are currently limited so discovery of new targets would be highly beneficial to patients. We examined the C-type lectin receptor CD302 as a potential therapeutic target for AML due to its selective expression in myeloid immune populations. In a cohort of 33 AML patients with varied morphological and karyotypic classifications, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population enriched with leukemic stem cells. A mAb targeting human CD302 was effective in mediating antibody dependent cell cytotoxicity and was internalised, making it amenable to toxin conjugation. Targeting CD302 with antibody limited in vivo engraftment of the leukemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. Expression was however found on the surface of haematopoietic stem cells suggesting that targeting CD302 would be most effective prior to haematopoietic transplantation. These studies provide the foundation for examining CD302 as a potential therapeutic target for AML.

Understanding the Journey of Human Hematopoietic Stem Cell Development.

Hematopoietic stem cells (HSCs) surface during embryogenesis leading to the genesis of the hematopoietic system, which is vital for immune function, homeostasis balance, and inflammatory responses in the human body. Hematopoiesis is the process of blood cell formation, which initiates from hematopoietic stem/progenitor cells (HSPCs) and is responsible for the generation of all adult blood cells. With their self-renewing and pluripotent properties, human pluripotent stem cells (hPSCs) provide an unprecedented opportunity to create in vitro models of differentiation that will revolutionize our understanding of human development, especially of the human blood system. The utilization of hPSCs provides newfound approaches for studying the origins of human blood cell diseases and generating progenitor populations for cell-based treatments. Current shortages in our knowledge of adult HSCs and the molecular mechanisms that control hematopoietic development in physiological and pathological conditions can be resolved with better understanding of the regulatory networks involved in hematopoiesis, their impact on gene expression, and further enhance our ability to develop novel strategies of clinical importance. In this review, we delve into the recent advances in the understanding of the various cellular and molecular pathways that lead to blood development from hPSCs and examine the current knowledge of human hematopoietic development. We also review how in vitro differentiation of hPSCs can undergo hematopoietic transition and specification, including major subtypes, and consider techniques and protocols that facilitate the generation of hematopoietic stem cells.

Mesenchymal stem cells-curcumin loaded chitosan nanoparticles hybrid vectors for tumor-tropic therapy

The combination of controlled release technology and targeted drug delivery has become a promising strategy for cancer therapy. In this study, cell-nanoparticle hybrid vector was constructed using mesenchymal stem cells as the targeting cellular carrier and biotinylated chitosan polymer nanoparticles as the drug depot. Drug-loaded nanoparticles (hydrodynamic size =377.0 ± 14.6 nm and zeta potential = 9.6 ± 1.9 mV) were prepared by encapsulating hydrophobic model drug curcumin into biotinylated chitosan polymer. The biotin-modified nanoparticles were anchored on biotinylated mesenchymal stem cells surface by biotin-avidin binding, achieving an upload of 54.73 ± 3.95 pg/cell. The anchorage of nanoparticles on mesenchymal stem cells had no effect on their viability and homing property. Biotin-avidin binding lasted over 48 h, which could be sufficient for cell-directed tumor-tropic delivery. The in vitro and in vivo anti-tumor results advocate that cell-nanoparticle hybrid vector could prove beneficial in pulmonary melanoma metastasis therapy.

Comparative analysis of FBS containing media and serum free chemically defined media for adipose derived stem cells production

Background & Aim For the successful development of production effective stem cell therapy, mass production of consistent quality cells is required. Excellent culture medium is important in mass production of quality cells. Classically, fetal bovine serum (FBS) has been used as culture supplement for stem cells. However, due to the undefined and heterologous composition of FBS, researchers have transitioned to more chemically defined serum-free media. In this study, we aimed to investigate and compare the stem cell characteristics cultured in both media as well as media performance tests. Methods, Results & Conclusion Methods Media performance was compared with cell growth rate, accumulated cell number, population homogeneity and cell viability. Comparative analysis of adipose derived mesenchymal stem cells (ADSCs) was performed based on stem cell surface marker expression, differentiation potency, genetic stability, senescence analysis, mRNA expression change, and protein expression change. Results Culture using SFCDM provided more stable population doubling time (PDT), more homogenous cell population, and more cells in a shorter time compared to FBS containing media (FBSCM). The cultured ADSCs were shown to be positive for CD73, CD90, and CD105, but negative for CD14, CD34, CD45, and HLA-DR in both FBSCM and SFCDM. Adipogenic differentiation capability was similar in both media groups, but chondrogenic and osteogenc differentiation capabilities of ADSCs cultured in SFCDM were higher than those cultured in FBSCM. ADSCs positive for β-galactosidase were about 3 times higher in FBSCM . These results indicate that culture in SFCDM has an advantage in reducing cellular senescence of ADSCs compared to FBS media. ADSCs cultured in FBSCM showed a significant increase in the formation frequency of nucleoplasmic bridges comparable to those cultured in SFCDM, suggesting that culture using SFCDM is more genetically stable. The mRNA expression levels of ADSCs cultured in FBSCM were significantly increased for KEGG classification of aging, apoptotic process, cell death, immune response and inflammatory response compared to SFCDM. Conclusion SFCDM can provide higher-speed cell production rate than the classical FBSCM. SFCDM enables the maintenance of higher population homogeneity, genetic stability, and excellent differentiation potency. Taken together, these results suggest that culture using SFCDM provides various advantages through which it is possible to obtain safer, stable, and larger amounts of ADSCs.

COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM IN CULTURE MEDIA FOR HUMAN DENTAL PULP STEM CELL PROLIFERATION

Objective: Ex vivo and in vitro cell cultures require a basal medium with added supplements containing growth factors, proteins, and enzymes tosupport attachment, growth, and proliferation. Fetal bovine serum (FBS) is used to supplement cell culture media. However, human platelet lysate(hPL) represents an attractive alternative as it is nonxenogeneic.Methods: Human third molars were collected from six healthy donors (19–35 years old) with no history of regular alcohol consumption or smoking.Human dental pulp stem cells (hDPSCs) at the second passage were divided into two culture media groups, 10% FBS and 5% hPL, as well as a controlgroup after 24 h of serum starvation. A flow cytometry analysis was conducted to measure CD90, CD105, CD73, CD34, CD45, and Human LeukocyteAntigen-DR isotype (HLA-DR). Cellular proliferation was evaluated on days 1, 3, and 5.Results: The flow cytometry analysis revealed that the majority of the cells expressed positive mesenchymal stem cell surface markers, includingCD73 (98.5%), CD90 (98.3%), and CD105 (71.0%), and lacked CD34, CD45, and HLA-DR. There were significant differences among the 5% hPL, 10%FBS, and control groups on days 1, 3, and 5.Conclusion: For a nonxenogeneic culture, 5% hPL can be used as an alternative in culture media for hDPSC proliferation.

A novel protocol for isolation and culture of multipotent progenitor cells from human urine

SummaryCell therapy holds promise for treating a variety of diseases. Seeking available source of adult stem cells remains a great challenge in cell therapy. Urine is considered as an ideal source of adult stem cells which can be easily acquired by noninvasive methods. However, specific cell types in urine have not been well documented. Here, the aim of our study is to identify cell types in urine, and isolate and expand progenitor/stem cells from human urine and further evaluate their multipotency.Urine samples were collected from healthy donors. The cell suspension was seeded and selected because of plastic adherence. Colonies with two different morphologies appeared 7 days later. One type of colony was spindle-shaped and fibroblast-like; the other cell type displayed rounder shape. Cells that displayed fibroblast-like shape were selectively enriched using a cloning cylinder. Then multidifferentiation induction assays and immunophenotyping assays were applied. Characterization assays indicated that adherent cells possessed potent trilineage differentiation capacity and expressed CXCR4 and Nanog, as well as some mesenchymal stem cell surface antigens (including CD90 and CD44). Taken together, at least two cell populations exist in human urine. A stem cell subpopulation with trilineage differentiation capacity from human urine can be selectively enriched using the cloning cylinder method. Urine may become an ideal source of adult stem cells for cell therapy and further clinical implications.

Sulforaphane Suppresses the Growth of Triple-negative Breast Cancer Stem-like Cells In vitro and In vivo.

Triple-negative breast cancer (TNBC) represents the poorest prognosis among all of breast cancer subtypes with no currently available effective therapy. In this study, we hypothesized that sulforaphane, a dietary component abundant in broccoli and its sprouts, can inhibit malignant cell proliferation and tumor sphere formation of cancer stem-like cells (CSC) in TNBC. CSC population was isolated using FACS analysis with the combined stem cell surface markers, CD44+/CD24-/CD49f+ The effect of sulforaphane on a stem-related embryonic oncogene CRIPTO-1/TDGF1 (CR1) was evaluated via ELISA. In vivo, BalbC/nude mice were supplemented with sulforaphane before and after TNBC cell inoculation (daily intraperitoneal injection of 50 mg sulforaphane/kg for 5 and 3 weeks, respectively), and the effects of sulforaphane during mammary tumor initiation and growth were accessed with NanoString gene analysis. We found that sulforaphane can inhibit cell proliferation and mammosphere formation of CSCs in TNBC. Further analysis of gene expression in these TNBC tumor cells revealed that sulforaphane significantly decreases the expression of cancer-specific CR1, CRIPTO-3/TDGF1P3 (CR3, a homologue of CR1), and various stem cell markers including Nanog, aldehyde dehydrogenase 1A1 (ALDH1A1), Wnt3, and Notch4. Our results suggest that sulforaphane may control the malignant proliferation of CSCs in TNBC via Cripto-mediated pathway by either suppressing its expression and/or by inhibiting Cripto/Alk4 protein complex formation. Thus, the use of sulforaphane for chemoprevention of TNBC is plausible and warrants further clinical evaluation.

Effect of CXCR9-CXCR3 Cytokine Signaling Pathway in Polycytemia Vera

<strong>Objective</strong>: Myeloproliferative neoplasms (MPNs) are among the most common myeloproliferative disorders. MPNs are characterized by excessive production of terminally differentiated blood cells. The expression of CXCR3, responsible for regulating the proliferation of healthy hematopoietic stem cells, is known to be induced by granulocyte-macrophage colony stimulating factor (GM-CSF). The aim of this study is to investigate the role of CXCL9-CXCR3 signaling pathway in the progression of MPN. <strong>Material and Method</strong>: We determined the expression of CXCL9 and two isoforms of the CXCR3 receptor (CXCR3A and CXCR3B) on the surface of human peripheral blood mononuclear cells (PBMC) and cancer stem cells. Real-time polymerase chain reaction (qPCR) was used to measure expression levels, and flow cytometry was used to examine the presence of cell surface receptors. In addition, qPCR was used to quantify CXCR3 expression after GM-CSF application in cell culture. In PBMC and CD34+ cells, the mRNA level of CXCR3A expression was found to be elevated in patients with MPN. <strong>Results</strong>: There was no statistically significant difference in mRNA expression of CXCR3B and CXCL9 between patients and healthy controls.There was significant reduction in cell surface expression of the CXCR3 receptor in PBMC obtained from patients. T <strong>Conclusion</strong>: hus, these results indicate that imbalance in the expression of CXCR3A/CXCR3B isoforms and chemokines CXCL9, CXCL10 and CXCL11 that bind to these receptors, may mediate inflammation and cancer progression in MPN.

Surface tethering of stem cells with H2O2-responsive anti-oxidizing colloidal particles for protection against oxidation-induced death

Mesenchymal stem cells are the new generation of medicine for treating numerous vascular diseases and tissue defects because of their ability to secrete therapeutic factors. Poor cellular survival in an oxidative diseased tissue, however, hinders the therapeutic efficacy. To this end, we hypothesized that tethering the surface of stem cells with colloidal particles capable of discharging antioxidant cargos in response to elevated levels of hydrogen peroxide (H2O2) would maintain survival and therapeutic activity of the stem cells. We examined this hypothesis by encapsulating epigallocatechin gallate (EGCG) and manganese oxide (MnO2) nanocatalysts into particles comprising poly(d,l-lactide-co-glycolide)-block-hyaluronic acid. The MnO2 nanocatalysts catalyzed the decomposition of H2O2 into oxygen gas, which increased the internal pressure of particles and accelerated the release of EGCG by 1.5-fold. Consequently, stem cells exhibited 1.2-fold higher metabolic activity and 2.8-fold higher secretion level of pro-angiogenic factor in sub-lethal H2O2 concentrations. These stem cells, in turn, performed a greater angiogenic potential with doubled number of newly formed mature blood vessels. We envisage that the results of this study will contribute to improving the therapeutic efficacy of a wide array of stem cells.

Construction of nano-scale cellular environments by coating a multilayer nanofilm on the surface of human induced pluripotent stem cells.

Interactions with peripheral environments, such as extracellular matrix (ECM) and other cells, and their balance play a crucial role in the maintenance of pluripotency and self-renewal of human pluripotent stem cells. In this study, we focused on a nano-sized artificial cellular environment that is directly attached to the cytoplasmic membrane as a facile method that can effect intercellular interactions at the single-cell level. We designed multilayered nanofilms that are self-assembled on the surface of human induced pluripotent stem cells (iPSCs), by repetitive adsorption of fibronectin and heparin or chondroitin sulfate. However, the surface modification process could also lead to the loss of cell-cell adhesion, which may result in apoptotic cell death. We investigated the proliferation and pluripotency of the iPSCs coated with the nanofilm in order to establish the suitable nanofilm structure and coating conditions. As a result, the cell viability reduced with the increase in the duration of the coating process, but the undifferentiated state and proliferation of the cells were maintained until 2 bilayers were coated. To suppress the dissociation-induced apoptosis, Y-27632, the Rho-associated kinase inhibitor (ROCKi), was added to the coating solution; this allowed the coating of up to 4 bilayers of the nanofilm onto the iPSCs. These results are expected to accelerate the pace of iPSC studies on 3-dimensional cultures and naïve pluripotency, in which the regulation of cellular interactions plays a critical role.

Enhanced Kat3A/Catenin transcription: a common mechanism of therapeutic resistance.

Cancers are heterogeneous at the cellular level. Cancer stem cells/tumor initiating cells (CSC/TIC) both initiate tumorigenesis and are responsible for therapeutic resistance and disease relapse. Elimination of CSC/TIC should therefore be able to reverse therapy resistance. In principle, this could be accomplished by either targeting cancer stem cell surface markers or “stemness” pathways. Although the successful therapeutic elimination of “cancer stemness” is a critical goal, it is complex in that it should be achieved without depletion of or increases in somatic mutations in normal tissue stem cell populations. In this perspective, we will discuss the prospects for this goal via pharmacologically targeting differential Kat3 coactivator/Catenin usage, a fundamental transcriptional control mechanism in stem cell biology.

PR1P ameliorates neurodegeneration through activation of VEGF signaling pathway and remodeling of the extracellular environment

Neurodegenerative diseases affect millions of people worldwide. Optic neuropathies are the most commonly occurring neurodegenerative diseases, characterized by progressive retinal ganglion cell (RGC) degeneration. We recently reported that Prominin-1, a protein found on the surface of stem cells, interacts with VEGF and enhances its activity. VEGF is known to have various protective roles in the nervous system. Subsequently, we have developed a 12-mer peptide derived from Prominin-1, named PR1P, and investigated its effects on neuronal survival of damaged RGCs in a rat model of optic nerve crush (ONC). PR1P prevented RGC apoptosis resulting in improvement of retinal function in the rat ONC model. PR1P treatment significantly increased phosphorylation of ERK and AKT and expression its downstream proteins c-fos and Egr-1 in the retina. Additionally, PR1P beneficially increased the MMP-9/TIMP-1 ratio and promoted glial activation in the retina of ONC rats. Thus, PR1P displayed neuroprotective effects through enhanced VEGF-driven neuronal survival and reconstruction of the extracellular environment in ONC model. Our data indicate that PR1P may be a promising new clinical candidate for the treatment of neurodegenerative diseases.

Targeting Cancer Stem Cells to Overcome Chemoresistance.

Cancers are heterogeneous at the cell level, and the mechanisms leading to cancer heterogeneity could be clonal evolution or cancer stem cells. Cancer stem cells are resistant to most anti-cancer treatments and could be preferential targets to reverse this resistance, either targeting stemness pathways or cancer stem cell surface markers. Gold nanoparticles have emerged as innovative tools, particularly for photo-thermal therapy since they can be excited by laser to induce hyperthermia. Gold nanoparticles can be functionalized with antibodies to specifically target cancer stem cells. Preclinical studies using photo-thermal therapy have demonstrated the feasibility of targeting chemo-resistant cancer cells to reverse clinical chemoresistance. Here, we review the data linking cancer stem cells and chemoresistance and discuss the way to target them to reverse resistance. We particularly focus on the use of functionalized gold nanoparticles in the treatment of chemo-resistant metastatic cancers.

Gadolinium Doping Enhances the Photoacoustic Signal of Synthetic Melanin Nanoparticles: A Dual Modality Contrast Agent for Stem Cell Imaging.

In this paper, we show that gadolinium-loaded synthetic melanin nanoparticles (Gd(III)-SMNPs) exhibit up to a 40-fold enhanced photoacoustic signal intensity relative to synthetic melanin alone and higher than other metal-chelated SMNPs. This property makes these materials useful as dual labeling agents because Gd(III)-SMNPs also behave as magnetic resonance imaging (MRI) contrast agents. As a proof-of-concept, we used these nanoparticles to label human mesenchymal stem cells. Cellular uptake was confirmed with bright-field optical and transmission electron microscopy. The Gd(III)-SMNP-labeled stem cells continued to express the stem cell surface markers CD73, CD90, and CD105 and proliferate. The labeled stem cells were subsequently injected intramyocardially in mice, and the tissue was observed by photoacoustic and MR imaging. We found that the photoacoustic signal increased as the cell number increased (R 2 = 0.96), indicating that such an approach could be employed to discriminate between stem cell populations with a limit of detection of 2.3 × 104 cells in in vitro tests. This multimodal photoacoustic/MRI approach combines the excellent temporal resolution of photoacoustics with the anatomic resolution of MRI.

The influence of two different cultivations on human liver cancer cell line SMMC-7721 cells

Objective To observe and compare thechange of human liver cancer cell line SMMC-7721 cells’ related characteristics using adherent cultivation and suspension cultivation. Methods (1) To collect human liver cancer cell line SMMC-7721 cells cultured with ordinary medium (adherent cultivation) and with serum-free medium (suspension cultivation). (2) To observe morphologic patterns of SMMC-7721 tumor cells treated with two kinds of cultivationunder inverted microscope. (3) To measure expressions of tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds of cultivation by the flow cytometry. (4) To detect mRNA expressions of the tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds cultivation by real-time quantitative polymerase chain reaction (Real-time PCR). (5) To observe changes of the tumor volume in nude mice after subcutaneous injection of SMMC-7721 tumor cells treated with two kinds of cultivation and to compare their tumorigenicity and multiplication capacity. Results Growing like typical cobblestone and arraying regularly for SMMC-7721 tumor cells in ordinary medium were observed under inverted microscope, while SMMC-7721 tumor cells in suspensive culture gathering from single cell into spherical or near spherical cells. The expression of CD90 and CD24 by the flow cytometryin suspension culture group was significantly higher than that in adherent culture group. The difference was statistically significant (CD90: P=0.000; CD24: P=0.000). But there was no significant difference between the expressions of CD133 in suspension culture group and adherent culture group. The Real-time PCR results showed that the mRNA expression of CD90 and CD24 in suspension culture group was significantly higher than that in adherent culture group. The difference was statistically significant (CD90: P=0.020, CD24: P=0.000). But there was no significant difference on expressions between suspension culture group and adherent culture group for the CD133. Statistical analysis of the tumorigenic ability and capacity to proliferate in vivo of tumor cells treated with two kinds of cultivation showed the suspension culture group’s capacity to proliferate in vivo was better than the adherent culture group. And tumorigenic ability in the suspension culture groups was higher than that in the adherent culture group. Conclusion For the human liver cancer cell line SMMC-7721 cells, hepatocarcinoma stem-like cells in suspension culture could be obtained more than that in adherent culture. Key words: Liver cancer stem-like cells; Suspension culture; SMMC-7721; CD90; CD133; CD24

Functional Profiling of Patient AML Stem Cells Reveals Mechanisms of Epigenetic Plasticity Controlling Therapy Resistance

Treatment of acute myeloid leukemia remains inadequate, largely due to our limited understanding of therapy resistance and lack of effective therapeutic targets. Recently, we and others have used functional genomic and proteomic methods to elucidate the mechanisms of therapy resistance. These studies have revealed the existence of privileged populations of AML cells with distinct signaling properties. AML is known to have leukemia stem cells (LSCs), which exhibit self-renewal and cell-cycle quiescence, and contribute to chemotherapy resistance. However, their prospective isolation has been hindered by their variable immunophenotypes, constituting an important gap to understanding their biology, as a prelude to developing improved therapies.Here, we used chemical protein labeling to prospectively isolate AML quiescent and stem cells from primary patient leukemias, based on proteome production and renewal. First, we developed optimized protein-labeling conditions to preserve stem cell function, as demonstrated by transplantation of labeled mouse hematopoietic stem cells into lethally irradiated recipients. We applied this method to prospectively isolate primary patient AML quiescent cells upon transplanting labeled specimens into immunodeficient NSG mice. These studies showed that label-retaining, but not their label non-retaining populations, are comprised by AML stem cells, as evidenced by serial transplantation and limiting dilution (Figure 1). Using genome sequencing of label-retaining and non-retaining populations, we confirmed the absence of genetic mutations associated with AML proteome quiescence, consistent with their epigenetic basis. To confirm this epigenetic mechanism directly, we performed re-labeling experiments, observing reversible development of quiescent stem cells from their proliferating counterparts. To define the epigenetic mechanisms of AML quiescence, we used assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and mRNA sequencing (RNA-seq) of label-retaining versus non-retaining populations from three different therapy-resistant MLL-, MOZ-, and NUP98-rearranged patient leukemias. Gene set enrichment analysis demonstrated significant association with reported gene expression programs associated with hematopoietic and AML stem cell function, and FACS analysis showed the presence of some but not all reported stem cell surface markers, including CD34, CD38, CD117, CD90, CD45RA, and CD123. Remarkably, in spite of the biological differences among these subtypes, we observed a common set of genes and differentially accessible chromatin loci in AML quiescent as compared to proliferating cells. Motif analysis revealed that differentially accessible chromatin in the label-retaining cell population was predominantly comprised by the E26 transformation-specific (ETS) family transcription factor (TF)-binding motifs. For example, the ETS-related gene (ERG)-binding motif covered more than 50% of differentially accessible chromatin in the label-retaining cell population (p = 10e-7). Consistently, label-retaining quiescent cells exhibited significant induction of gene expression programs associated with ETS transcription factor function.In summary, this work presents a functional approach for the prospective isolation of primary patient AML quiescent cells. Human AML quiescent cells constitute a distinct population with leukemia-initiating and self-renewal capacity, as well as epigenetic plasticity and reversible induction in diverse genetic disease subtypes. We anticipate that the reported gene expression and chromatin profiles, including those controlled by ETS transcription factors, should define their control mechanisms and targets for improved therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

4-Acetylantroquinonol B from antrodia cinnamomea enhances immune function of dendritic cells against liver cancer stem cells

The functions of 4-acetylantroquinonol B (4-AAQB), a ubiquinone derivative isolated from the mycelium of Antrodia cinnamomea, in immunotherapy for liver cancer were investigated. We found that 4-AAQB could inhibit liver cancer stem cell related manifestations and activate the antitumor ability of dendritic cells. Specifically, 4-AAQB can inhibit EpCAM, AFP and related pathways of HepG2 cells. It also significantly decreases the expression of β-catenin, inhibits the tumorigenicity and decreases the secretion of immune escape related cytokines. Moreover, 4-AAQB can stimulate the proliferation of immune cells and promote the endocytosis of immature dendritic cells. When co-cultured immature dendritic cells with EpCAM+ HepG2 cells, 4-AAQB enhanced the expression of MHC class I and II on the surface of liver cancer stem cells and dendritic cells, increased the expression of costimulatory molecules CD80 of dendritic cells and cytokines related to immune activation. In conclusion, 4-AAQB from Antrodia cinnamomea can enhance immune function of dendritic cells against liver cancer stem cells, and may have the potential to be used for liver cancer prevention and immunotherapy.