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Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types,and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described. View Publication

A two-step separation procedure is described for the positive selection of cells based on their reactivity with mouse monoclonal antibodies. In the first step cells are specifically cross-linked to hapten-modified glass beads using tetrameric monoclonal antibody complexes. In the second step bound cells are selectively eluted by reductive cleavage of the tetrameric antibody complexes. The latter are comprised of two mouse IgG1 monoclonal antibodies (one recognizing a cell surface antigen on target cells and the other a hapten coupled to the glass beads) bound together by two F(ab')2 fragments of rat anti-mouse IgG1 monoclonal antibody. The complexes provide a specific cleavable cross-link between cell and bead because the disulfide bonds between the two Fab' arms of the F(ab')2 fragments can be broken under relatively mild conditions using dithiothreitol. This specific cleavage of the cross-linker allows elution of the specifically absorbed cells without co-elution of non-specifically bound cells. This is shown in the purification of CD3+ T cells from human peripheral blood,where the removed fractions were over 90% pure and approximately 50% of the positive cells were recovered. Separation of cells labelled with limiting amounts of tetrameric antibody complexes demonstrated that this separation technique was also effective for the purification of cells expressing low amounts of antigens. This was confirmed by the purification of CD34-positive cells from human bone marrow. With this approach,colony-forming cells were enriched 15-24-fold over density separated marrow. View Publication

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons,leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS),among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations,usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies,however due to the differences between rodents and humans,it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore,we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,sod1 mutations,FUS,ANG and FIG4 mutations. Certain mutations are represented with more than one line,which allows for studies of variable genetic backgrounds. In addition,these iPSCs can be successfully differentiated to astroglia,a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View Publication

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs),but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs,binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7,which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database,we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore,we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together,we uncover a potential mechanism of how PTEN safeguards NSCs,and establish a cellular platform to identify factors involved in NSC transformation,potentially permitting personalized treatment of glioblastoma. View Publication

Although since 1998 more than 1,200 different hESC lines have been established worldwide,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups underrepresented among the currently available lines. The methodology of hESC derivation has evolved significantly since the initial derivations using human LIF (hLIF) for maintenance of pluripotency. However,there are still a number of alternative strategies for the different steps involved in establishing a new line of hESC. We have analyzed the different strategies/parameters used between 1998 and 2010 for the derivation of the 375 hESC lines able to form teratomas in immunocompromised mice deposited in two international stem cell registries. Here we describe some trends in the methodology for establishing hESC lines,discussing the developments in the field. Nevertheless,we describe a much greater heterogeneity of strategies for hESCs derivation than what is used for murine ESC lines,indicating that optimum conditions have not been identified yet,and thus,hESC establishment is still an evolving field of research. View Publication

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors,but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue,we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct,which inhibits signaling through all Notch receptors,and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease,but not a complete abrogation,of these cells in dnMAML-expressing cells. Interestingly,when assessed in secondary assays in vitro or in vivo,there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool,which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However,the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal,proliferation,and maintenance of embryonic stem cells (ESCs),but the contribution of this pathway or its well-known negative regulator,phosphatase,and tensin homolog deleted on chromosome ten (Pten),to somatic cell reprogramming remains largely unknown. Here,we show that activation of the PI3K pathway by the Pten inhibitor,dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate,improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs. View Publication

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents,including histamine,serine proteases,and various eicosanoids and cytokines. As such,a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology,assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells,cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive,requiring only 500 cells per data point,which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation,to enable innovative drug discovery efforts to be prosecuted. View Publication

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay,we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms,we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFN$\gamma$ production. Moreover,TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I,adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings,we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis,acting as a regulator for the induction of different effector functions. View Publication

Recent studies suggest that highly activated,polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity,promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously,we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response,which mediated stronger antitumor immunity in vivo. In this study,the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition,by using T-cell depletion,isolation,and adoptive T-cell therapy,it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells,promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-? and enhanced the cytotoxic effector function of NK cells and CD8+ T cells,and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-?. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory,we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application. View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication