High-efficient serum-free differentiation of endothelial cells from human iPS cells.

INTRODUCTION: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. METHOD: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. RESULT: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). CONCLUSION: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels.

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell-cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testingin vitro(organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapiesin vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells inpoly-N-isopropylacrylamid(PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Scalable Generation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cells (hiPSCs) can be expanded at limitless scale in vitro and give rise to various organotypic cells, cardiomyocytes (CMs) among them. Advanced protocols shape the differentiation process of pluripotent stem cells by controlled growth factor application. Modulating the Wnt signaling pathway is effective to direct hiPSCs to CMs (hiPSC-CMs) and native growth factors were replaced by small chemical compounds. Here, we describe a refined protocol for scalable generation of hiPSC-CMs that manipulates porcupine and tankyrase sub-pathways of Wnt signaling for tight inhibition of non-canonical Wnt signaling. The approach results in a differentiation efficiency toward hiPSC-CMs of 87 ± 0.9% in stirred bioreactor cultures and yields about 70 million hiPSC-CMs per 100 mL serum free cardiac differentiation medium. The differentiation protocol is easily adapted from 3D to 2D culture and vice versa and has been demonstrated to work with different hiPSC lines.

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations.

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.

A teaching tool about the fickle p value and other statistical principles based on real-life data.

A poor understanding of statistical analysis has been proposed as a key reason for lack of replicability of many studies in experimental biomedicine. While several authors have demonstrated the fickleness of calculated p values based on simulations, we have experienced that such simulations are difficult to understand for many biomedical scientists and often do not lead to a sound understanding of the role of variability between random samples in statistical analysis. Therefore, we as trainees and trainers in a course of statistics for biomedical scientists have used real data from a large published study to develop a tool that allows scientists to directly experience the fickleness of p values. A tool based on a commonly used software package was developed that allows using random samples from real data. The tool is described and together with the underlying database is made available. The tool has been tested successfully in multiple other groups of biomedical scientists. It can also let trainees experience the impact of randomness, sample sizes and choice of specific statistical test on measured p values. We propose that live exercises based on real data will be more impactful in the training of biomedical scientists on statistical concepts.

Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are promising candidates to treat myocardial infarction and other cardiac diseases. Such treatments require pure cardiomyocytes (CMs) in large quantities. Methods: In the present study we describe an improved protocol for production of hiPSC-CMs in which hiPSCs are first converted into mesodermal cells by stimulation of wingless (Wnt) signaling using CHIR99021, which are then further differentiated into CM progenitors by simultaneous inhibition of porcupine and tankyrase pathways using IWP2 and XAV939 under continuous supplementation of ascorbate during the entire differentiation procedure. Results: The protocol resulted in reproducible generation of >90% cardiac troponin T (TNNT2)-positive cells containing highly organized sarcomeres. In 2D monolayer cultures CM yields amounted to 0.5 million cells per cm2 growth area, and on average 72 million cells per 100 mL bioreactor suspension culture without continuous perfusion. The differentiation efficiency was hardly affected by the initial seeding density of undifferentiated hiPSCs. Furthermore, batch-to-batch variations were reduced by combinatorial use of ascorbate, IWP2, and XAV939. Conclusion: Combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling and continuous ascorbate supplementation, enable robust and efficient production of hiPSC-CMs.

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

Acid-Sensitive Ion Channels Are Expressed in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are potential sources for cardiac regeneration and drug development. hiPSC-CMs express all the cardiac ion channels and the unique cardiac Ca2+-signaling phenotype. In this study, we tested for expression of acid sensing ion channels (ASICs) in spontaneously beating cardiomyocytes derived from three different hiPSC lines (IMR-90, iPSC-K3, and Ukki011-A). Rapid application of solutions buffered at pH 6.7, 6.0, or 5.0 triggered rapidly activating and slowly inactivating voltage-independent inward current that reversed at voltages positive to ENa, was suppressed by 5 µM amiloride and withdrawal of [Na+]o, like neuronal ASIC currents. ASIC currents were expressed at much lower percentages and densities in undifferentiated hiPSC and in dermal fibroblasts. ASIC1 mRNA and protein were measured in first 60 days but decreased in 100 days postdifferentiation hiPSC cultures. Hyperacidification (pH 5 and 6) also triggered large Ca2+ transients in intact hiPSC-CMs that were neither ruthenium red nor amiloride-sensitive, but were absent in whole cell-clamped hiPSC-CMs. Neither ASIC1 current nor its protein was detected in rat adult cardiomyocytes, but hyperacidification did activate smaller and slowly activating currents with drug sensitivity similar to TRPV channels. Considering ASIC expression in developing but not adult myocardium, a role in heart development is likely.