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Human microglia, as innate immune cells of the central nervous system (CNS), play a central role in the pathogenesis of a large number of neurological and psychiatric disorders. However, experimental access to primary human microglia for biomedical applications such as disease modeling is extremely limited. While induced pluripotent stem cells (iPSCs) could provide an alternative source of microglia, the reenactment of their complex ontogenesis with a yolk sac origin and subsequent priming upon CNS invasion has remained a challenge. Here, we report a developmentally informed in vitro differentiation method for large-scale production and cryopreservation of iPSC-derived microglia (iPSdMiG). Specifically, iPSCs were propagated in conditions yielding both yolk sac hematopoietic derivatives and early neuroepithelial cells. To enable large-scale production, we implemented 3D bioreactor-based dynamic culture conditions and the use of novel mesh macrocarriers. Under these conditions, microglia could be harvested across a time period of at least 6 weeks, with 1 × 106 iPSCs giving rise to up to 45 × 106 iPSdMiG. The transcriptomic profile of iPSdMiG showed high similarity to adult human microglia, and harvested cells were immunopositive for typical microglial markers. In addition, iPSdMiG were able to secrete pro-inflammatory cytokines, engaged in phagocytotic activity, produced reactive oxygen species and lent themselves to co-culture studies in neural 2D and 3D systems. Importantly, iPSdMiG were efficiently cryopreserved, enabling the establishment of donor-specific microglia cell banks for disease modeling, drug discovery and eventually cell therapy. Graphical abstract Main points. Scalable generation of iPSC-derived multi-lineage embryoid bodies on macrocarriers, reproducibly releasing microglia exhibiting characteristic markers and function. Cells are transcriptomically similar to primary human microglia and cryopreservable.

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. Electronic supplementary material The online version of this article (doi:10.1007/s12015-014-9564-6) contains supplementary material, which is available to authorized users.

Stem cells have become a major focus of scientific interest as a potential source of somatic cell types for biomedical applications. Understanding and controlling the elicitors and mechanisms in differentiation of pluripotent stem cell-derived somatic cell types remains a key challenge. The major types of molecular processes that control cellular differentiation involve evolutionary conserved cell signaling pathways. Notch receptors participate in a wide variety of biological processes, including cell fate decisions of stem cells. This study explores the potential of protein transduction to directly deliver recombinant Notch-1 intracellular domain (NICD) into mammalian cells in order to accomplish transgene-free Notch activation. We engineered a cell-permeant version of NICD and explored its function on mouse and human neural stem cells. We show that NICD transduction modulates known direct and indirect Notch target genes and antagonizes the DAPT-mediated inhibition of Notch signaling on the transcriptional level. Moreover, NICD enhances cell proliferation accompanied by increased cyclin D1 and decreased p27 protein levels. In the absence of growth factors NICD strongly impairs neuronal differentiation while being insufficient to keep cells in a proliferative state. Furthermore, our studies depict NICD protein transduction as a novel tool for a time and dose-dependent non-genetic modulation of Notch signaling to decipher its cellular functions.

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 +/- 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.

The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.