Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5 -knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

Primary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients.