技术资料

View Publication

To gain insight into the molecular regulation of human heart development,a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal,adult,and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific,transitional cardiac specification,and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless,analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1,miR-133a/b,and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together,these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. View Publication

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation,and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and,with quantitative cell division tracking and fate monitoring,identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion,during directed differentiation,to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs),share the properties of unlimited self-renewal and the capacity to become any cell type in the body,making them well suited for regenerative medicine and cell therapy. So far,almost all hPSC lines have been directly or indirectly exposed to animal-derived products,which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation,propagation,and cryopreservation of hPSCs. Moreover,the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined,xeno-free conditions,2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium,matrix,and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor,Rho-associated kinase inhibitor (ROCK),and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system,and not at all in another. In this study,we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined,xeno-free conditions. View Publication

A method for the generation of human induced pluripotent stem (iPS) cells was established. This method employs adenovirus carrying the ecotropic retrovirus receptor mCAT1 and Moloney murine leukemia virus (MMLV)-based retroviral vectors carrying the four transcription factors POU5F1 (OCT3/4),KLF4,SOX2,and MYC (c-Myc) (Masaki H & Ishikawa T Stem Cell Res 1:105-15,2007). The differentiation of human iPS cells into hepatic cells was performed by a stepwise protocol (Song Z et al. Cell Res 19:1233-42,2009). These cells have potential as patient-specific in vitro models for studying disease etiology and could be used in drug discovery programs tailored to deal with genetic variations in drug efficacy and toxicity. View Publication

Triple-negative breast cancer (TNBC) is an aggressive and clinically challenging subtype defined by the absence of estrogen receptor,progesterone receptor,and HER2 amplification,resulting in poor prognosis and limited therapeutic options. Targeting alternative molecular pathways is urgently needed to overcome resistance and improve patient outcomes. CD147 has emerged as surface marker associated with tumor progression and immune evasion. In this study,CD147 and MHC class I—a key inhibitory ligand for natural killer cells—were analyzed in breast cancer cell lines (MCF7,MDA-MB-453,MDA-MB-231,and HCC38) using flow cytometry. The therapeutic efficacy of a humanized anti-CD147 monoclonal antibody (HuM6-1B9) was evaluated for its capacity to potentiate antibody-dependent cellular cytotoxicity (ADCC). HuM6-1B9 demonstrated the strong binding to MDA-MB-231 (KD = 4.982 nM) and HCC38 (KD = 4.523 nM),which are representative TNBC cell lines. In 3D spheroid models,HuM6-1B9 significantly enhanced PBMC-mediated ADCC,leading to a marked reduction in TNBC spheroid viability. Co-culture of CFSE-labeled MDA-MB-231 and HCC38 cells with primary NK cells confirmed robust ADCC,achieving 50% and 70% cytotoxicity,respectively,despite high MHC class I expression. Live-cell imaging demonstrated caspase-3/7 activation consistent with apoptosis in NK-targeted tumor cells,while CD107a degranulation and IFN-γ secretion confirmed the functional contribution of HuM6-1B9 to ADCC enhancement. Importantly,HuM6-1B9 did not promote migration or invasion in MDA-MB-231 cells,supporting its safety profile regarding metastasis. Collectively,these findings establish HuM6-1B9 as a promising immunotherapeutic candidate that overcomes immune resistance and selectively eliminates TNBC cells through ADCC without enhancing metastatic potential. By integrating mechanistic assays of NK cytotoxicity,apoptosis,and 3D tumor spheroids,this study provides clinically relevant insights underscoring the translational potential of HuM6-1B9 in TNBC immunotherapy. View Publication

EVI1-rearranged acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21q26) represents a distinct and aggressive subtype characterized by poor prognosis and limited treatment options. However,the optimal strategy to overcome resistance to conventional therapy remains elusive. Building upon observations correlating EVI1 overexpression with reduced sensitivity to venetoclax,a BH3-mimetic BCL-2 inhibitor,we investigated the mechanisms of resistance to venetoclax in combination with hypomethylating agents in inv(3)/t(3;3) AML cells. Utilizing novel murine models recapitulating inv(3) AML with concomitant SF3B1 mutations,we conducted comprehensive phenotypic and transcriptomic analyses in the presence or absence of venetoclax-containing therapy. Despite initial therapeutic responses,manifested as partially prolonged survival and myeloid differentiation,resistant leukemic cells demonstrated enhanced dependency on BRD4 and MYB pathways with a dormant phenotype. Notably,inhibition of either BRD4 or MYB significantly augmented the efficacy of venetoclax and hypomethylating agents in both murine and patient-derived AML models harboring inv(3) and SF3B1 mutations. These findings elucidate the transcriptional dynamics underlying venetoclax resistance and propose alternative therapeutic strategies targeting BRD4 and MYB as promising avenues for improving outcomes in patients with EVI1-rearranged AML. Our work highlights the necessity for innovative combination therapies to address the multifaceted mechanisms of resistance in this high-risk leukemia subtype. View Publication

Chagas disease,caused by the parasite Trypanosoma cruzi,affects millions of people globally. Unfortunately,the available treatment options,especially for the chronic stage of the disease,are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite,there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here,we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi,we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover,their human origin,stable genetic background,and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi,the causative agent of Chagas disease,is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease,often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date,only benznidazole and nifurtimox are used for treatment of the infection,but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials,causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns. View Publication

Pluripotent cancer stem cells play a pivotal role in inducing phenotypic plasticity across various cancer types,including bladder cancer. This plasticity,crucial for cancer progression,is largely regulated by epigenetic modifications including N6-methyladenosine (m6A) in RNAs. However,the role of the m6A reader protein YTHDC2 in this process remains poorly understood. In this study,we uncovered that the depletion of YTHDC2 significantly increased the pool of bladder cancer stem cells (BCSCs),resulting in a phenotypic shift towards a more invasive subtype of bladder cancer. This shift was characterized by enhanced proliferation,migration,invasion,and self-renewal capabilities of cancer cells,highlighting YTHDC2’s function as a tumor suppressor. Mechanistically,YTHDC2 recognized and bound to m6A-modified SOX2 mRNA,resulting in translational inhibition of SOX2. In conclusion,our study identifies YTHDC2 as a tumor suppressor in bladder cancer through inhibiting SOX2-mediated cell pluripotency and underscores the therapeutic potential of targeting the YTHDC2-SOX2 axis in bladder cancer. View Publication