Stem Cell Research

The purpose of this study was to optimize the efficiency and cost of human gastruloid formation by testing and adjusting individual parameters using as examples two distinct human pluripotent stem cell lines, both available from the UK Stem Cell Bank. For the first step, commercially sourced culture medium was replaced with a home-made defined recipe, known as N2B27, into which specific reagents can be titrated. By reducing the concentration of Activin A to 15% of the original protocol, efficient elongation of aggregated embryoid bodies was achieved. Also, titrating initial cell density and delaying the brief culture in GSK3 inhibitor until the onset of cell aggregation in individual wells was advantageous. Efficiency of formation of early gastruloids exhibiting the expected regionalization of the three embryonic germ layers was further enhanced by addition of TGF{beta}-inhibitor. The optimization steps presented here thus provide a simplified, robust and relatively economical protocol for consistent generation of elongated gastruloids from human pluripotent stem cells. This streamlined method improves accessibility and reproducibility, also providing a standardized platform to investigate fundamental principles of early human development. Summary StatementWe present an optimized protocol for human gastruloid production that should enhance efficiency, reproducibility and affordability for future in vitro studies into early human post-implantation development.

Intestinal organoids are promising tools in the context of animal experiment reduction. Yet, a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig, a valuable model of Humans. Organoids were derived from jejunal and colonic ISC obtained from 1, 7, 28, 36 and 180-day old pigs and passaged three times. We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (P<0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, P<0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (P<0.05), spheroids were larger during the primary culture but smaller after two passages (P<0.05) and organoids smaller after one passage (P>0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) were observed between organoids originating from 7 or 180-day old pigs, while difference between intestinal site origin were noticed. In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence their phenotype.

Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways. MethodsWe previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data. ResultsCulture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGF{beta}, and NF{kappa}B pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells. ConclusionResults support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGF{beta}, Notch, and NF{kappa}B, and low Wnt pathway activity scores. Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

Given the considerable interest in using stem cells for modelling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), play a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that metabolic switch from glycolysis to OXPHOS is germ-layer specific as during early ectoderm commitment, glycolysis remains active while during the transition to mesoderm and endoderm lineages, it is downregulated. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remains unclear. Here we address the question of mitochondrial adaption by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to functional cardiomyocytes. Contrary to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite clear mitochondrial remodeling giving increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work both highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptions upon further differentiation. Our results improve understanding of how mitochondria remodeling and the metabolism interact during differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content. Summary statementWe found that mitochondrial content decreases continuously during mesoderm differentiation, despite clear mitochondrial remodeling giving increased mitochondrial activity and higher levels of ATP-linked respiration during mesoderm differentiation.

It is still unclear if immune responses will compromise the large scale utilization of cell therapies derived from human induced pluripotent stem cells (hiPSCs). To answer this question, we used humanized mouse models and evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were rejected, engraftment of autologous cells was tolerated, indicating reprogramming and differentiation procedures are not immunogenic. We also demonstrated that hiPSC-derived myogenic progenitors, in opposition to hiPSCs, are not targeted by natural killer (NK) cells both in vitro and in vivo. Yet, adoptive transfer of NK cells can prevent the formation of hiPSC-derived teratoma. Overall, our findings suggest that hiPSC-derived muscular therapies will be tolerated in presence of a competent human immune system and highlight the risk of forming a teratoma if using partially differentiated autologous human cells. HighlightsO_LIhiPSC-derived myofibers are tolerated in autologous humanized mouse models C_LIO_LIInfiltration of autologous T cells is not predictive of successful skeletal muscle engraftment C_LIO_LIAdoptive transfer of NK cells prevents the formation of hiPSCs derived teratomas C_LIO_LINK cells are unable to reject established teratomas C_LI

In vitro stem cell culture is demanding in terms of manpower and media supplements. In recent years, new protocols have been developed to expand pluripotent embryonic stem cells in suspension culture, which greatly simplifies cell handling and scalability. However, it is still unclear how suspension culture protocols with different supplements affect pluripotency, cell homogeneity and cell differentiation compared to established adherent culture methods. Here we tested four different culture conditions for mouse embryonic stem cells (mESC) and quantified chimerism and germ line transmission as well as in vitro differentiation into three-dimensional neuro-epithelia. We found that suspension culture supplemented with CHIR99021/LIF offers the best compromise between culturing effort, robust pluripotency and cell homogeneity. Our work provides a guideline for simplifying mESC culture and should encourage more cell biology labs to use stem cell-based organoids as model systems.

A complete knockout (KO) of a single key pluripotency gene has been shown to drastically affect embryonic stem cell (ESC) function and epigenetic reprogramming. However, knockin (KI)/KO of a reporter gene only in one of two alleles in a single pluripotency gene is considered harmless and is largely used in the stem cell field. Here, we sought to understand the impact of simultaneous elimination of a single allele in two ESC key genes on pluripotency potential and acquisition. We established multiple pluripotency systems harboring KI/KO in a single allele of two different pluripotency genes (i.e. Nanog+/-; Sall4+/-, Nanog+/-; Utf1+/-, Nanog+/-; Esrrb+/- and Sox2+/-; Sall4+/-). Interestingly, although these double heterozygous mutant lines maintain their stemness and contribute to chimeras equally to their parental control cells, fibroblasts derived from these systems show a significant reduction in their capability to induce pluripotency either by Oct4, Sox2, Klf4 and Myc (OSKM) or by nuclear transfer (NT). Tracing the expression of Sall4 and Nanog, as representative key pluripotency targeted genes, at early phases of reprogramming could not explain the seen delay/blockage. Further exploration identifies abnormal methylation landscape around pluripotent and developmental genes in the double heterozygous mutant fibroblasts. Accordingly, treatment with 5-azacytidine two days prior to transgene induction rescues the reprogramming defects. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction and suggests that insufficient levels of key pluripotency genes leads to DNA methylation abnormalities in the derived-somatic cells later on in development.

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here we explore requirement for the downstream signalling transducer and activator of transcription, STAT3 and its target, TFCP2L1, in maintenance of naive pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our novel image analysis tool, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1, underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, implicating involvement of signals in addition to LIF/IL6 for sustaining naive pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. Summary StatementInducing diapause in mouse embryos demonstrates that STAT3 and TFCP2L1 are essential for self-renewal of the epiblast, but only TFCP2L1 is required for derivation of embryonic stem cells.

By aggregating low numbers of mouse embryonic stem cells (mESCs) followed by exposure to Wnt activation, structures termed gastruloids are produced that display an anteroposterior organisation of cell types derived from all three germ layers. Since current gastruloid protocols display considerable heterogeneity between experiments in terms of morphology, elongation efficiency, and cell type composition, we investigated whether an optimised mESC pluripotency state would provide more consistent results. By growing mESCs in different intervals of ESLIF and 2i medium the pluripotency state of cells was modulated, and mESC culture as well as the resulting gastruloids were analysed. Microscopy analysis showed a pre-culture-specific effect on gastruloid formation, in terms of efficiency, elongation index and reproducibility. RNA-seq analysis of the mESC start population confirmed that short-term pulses of 2i and ESLIF modulate the pluripotency state, and result in different cellular states. With multiple epigenetic regulators among the top differentially expressed genes we further analysed genome-wide DNA methylation and H3K27me3 distributions. We observed the same divergence between conditions, most dominantly displayed in the promoter regions of developmental regulators. Lastly, when we investigated the cell type composition of gastruloids grown from these different pre-cultures, we observed that mESCs subjected to 2i-ESLIF preceding aggregation generated gastruloids more consistently, including more complex mesodermal contributions as compared to the ESLIF-only control. This indicates that optimisation of the mESCs pluripotency state allows the modulation of cell differentiation during gastruloid formation.

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic integrity in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based test for the detection of hotspot mutations test. Cell-line identity was authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by qPCR for progenitor, neuronal, cortical, and glial markers. Taken together, we present a multistep quality control workflow to evaluate variability and reproducibility across and between iPSCs.

BackgroundThe anthracycline doxorubicin (Dox) is a widely used genotoxic chemotherapeutic drug with known dose-limiting cardiotoxic effects. How Dox-induced damage either to cardiomyocytes or to cardiac stem cells, which may compromise cardiac regeneration, contributes to cardiotoxicity remains poorly understood. MethodsHere we used a human induced pluripotent stem cell (iPSC)-based model system to determine the sensitivity of stem cells (iPSCs) and iPSC-derived derived cardiomyocytes (iCMs) applying different treatment regimens of Dox. Next to a broad range of methods to determine cellular and mitochondrial functions we performed an in-depth whole genome transcriptome profiling in iPSCs as well as iCMs. ResultsAs compared to their differentiated counterparts, iPSCs are highly sensitive against even short pulse-treatments with low Dox concentrations. Using such rather mild treatment conditions, we observed major mitochondrial impairments as demonstrated by increased mitochondrial fragmentation, persistent loss of mitochondrial membrane potential, and reduced ATP levels, while neither a markedly increased nuclear DNA damage response nor apoptosis were detected. Albeit mitochondrial dysfunction was not accompanied by changes in mitochondrial ultrastructure or altered OXPHOS complex assembly, mitochondrial genome (mtDNA) organization was altered. This points to a possible role of mtDNA remodelling for contributing to the high susceptibility of iPSCs to Dox. Whole genome transcriptome profiling revealed major differences in the transcriptional response to Dox treatment between iPSCs and iCMs. We could show that a moderate and transient exposure of iPSCs to Dox is sufficient to cause major transcriptional changes as for example reflected by the downregulation of numerous pivotal genes regulating cellular homeostasis and energy metabolism in iPSCs. Furthermore, pulse-treatment with Dox at the iPSC stage, termed preconditioning here, shifts the global transcriptional landscape of iCMs towards the expression of genes associated with impaired cardiac muscle regeneration, disrupted energy metabolism, altered muscle contraction, and increased fibrosis. ConclusionsOur findings support the hypothesis that Dox-induced mitochondrial dysfunction and transcriptional preconditioning in stem cells results in an impaired regenerative capacity after differentiation. This highlights a potential critical role of stem cells in mediating Dox-induced cardiotoxicity.

SUMMARYCurrent protocols for the differentiation of cardiomyocytes from human induced pluripotent stem cells (iPSCs) generally require prolonged time in culture and result in heterogeneous cellular populations. We present a method for the generation of beating cardiomyocytes expressing specific ventricular markers after just 14 days. Addition of the pan-retinoic acid receptor inverse agonist BMS 493 to human iPSCs for the first 8 days of differentiation resulted in increased protein expression of the ventricular isoform of myosin regulatory light chain (MLC2V) from 18.7% {+/-} 1.72% to 55.8% {+/-} 11.4% (p <0.0001) in cells co-expressing the cardiac muscle protein troponin T (TNNT2). Increased MLC2V expression was also accompanied by a slower beating rate (49.4 {+/-} 1.53 vs. 93.0 {+/-} 2.81 beats per minute, p <0.0001) and increased contraction amplitude (201% {+/-} 8.33% vs. 100% {+/-} 10.85%, p <0.0001) compared to untreated cells. Improved directed differentiation will improve in vitro cardiac modeling.

Clinical use of human embryonic stem cells (ESCs) as a raw material requires good manufacturing practice-compliant axillary materials such as culture medium. To this end, animal components should not be used and contamination of virus/bacteria/fungus and allergens are a concern. In addition, animal components such as albumin and fetal bovine serum pose difficulties such as a lot-to-lot variation. However, only a limited number of animal component-free media have been developed to date. In this study, we investigated whether SEES2 ESCs can be stably propagated for 16 passages (54 population doublings) over a period of 60 days in a newly established Stem-Partner(R) ACF medium. SEES2 ESC maintained their intact karyotype, i.e. 46,XX, and their undifferentiated phenotypes after long-term culture. An in vitro differentiation assay revealed that SEES2 ESCs exhibited multipotency, i.e. endodermal, mesodermal and ectodermal differentiation. Subcutaneous implantation of SEES2 ESCs generated mature teratomas without malignant transformation. These results show that SEES2 ESCs in the Stem-Partner(R) ACF medium can be used to establish master cell banks for future regenerative medicine as well as other ESCs in the previously reported culture medium.

BackgroundThe utilization of stem cells (SCs) has led the way into a new era of therapeutics known as regenerative medicine. Their renewal property offers exciting possibilities in reversing tissue damage caused by metabolic and degenerative changes. Research should be conducted increasingly to explore the possibilities of SC utilization in Pakistan. ObjectivesTo assess the level of knowledge, perception, and attitude of medical students regarding stem cell research (SCR) and its application, to obtain a better insight into the future of stem cell therapy in Pakistan as it is a rapidly emerging field in medicine. Materials and MethodsThis cross-sectional study was carried out using a self-administered questionnaire filled by 206 medical students from different medical colleges in Pakistan. A convenience sampling method was used. Knowledge and attitude scores were calculated based on answers to 28 well-structured questions. Data was analyzed using SPSS. ResultsThe mean values of the answers showed that 60.2% (n=124) of the students had a good knowledge and 39.8% (n=82) had poor knowledge of stem cells. Whereas, 56.8% (n=117) expressed a positive attitude and 43.2% (n=89) expressed a negative attitude towards SCR. Independent t-test applied on knowledge score and attitude showed that the mean knowledge score of people with a positive attitude is higher i.e. 21.25 as compared to the mean knowledge score of people with negative attitude i.e. 19.21. And the difference of the means is significant at p=0.007. Thus, the attitude of students was observed to be significantly dependent on their knowledge about SCR. ConclusionThe results show that medical students have baseline knowledge about SC therapy and a positive attitude towards it. Seminars, workshops should be conducted and this topic should be added to their syllabus so that they obtain proper information about SCR and encourage further research.

Background & AimsHypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces inflammatory hypoxia, a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. MethodshISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1 response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. ResultsThe novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1 at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins ConclusionsHypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

Primary tissue stem cells are useful not only for basic cell biological research but also for therapeutic applications: however, their broader utility is often limited by technical challenges, such as a low efficiency of exogenous gene expression. PIEZO1 is a large mechanosensitive ion channel that plays an important role in muscle-resident stem cells, known as muscle satellite cells (MuSCs), during muscle regeneration. In this study, we developed a method for the ectopic expression of PIEZO1 in isolated MuSCs. Using a baculovirus vector system, we expressed PIEZO1 in myoblast C2C12 cells. Following optimization of the infection condition, we achieved robust PIEZO1 expression in isolated MuSCs during activated and differentiated states, with appropriate subcellular localization and ion channel activity. Importantly, the baculovirus-mediated PIEZO1 expression restored the reduced proliferative capacity of Piezo1-deficient MuSCs to a level comparable to wild-type cells, indicating that the exogenously expressed PIEZO1 is functionally equivalent to the endogenous protein. Overall, we established an efficient method for the transfer of the Piezo1 gene into isolated MuSCs, which should provide a versatile platform to study other large proteins in MuSCs. Summary StatementA baculovirus-based method was developed, enabling robust and functional PIEZO1 expression in isolated muscle satellite cells and providing a platform to study large proteins in isolated stem cells.

BackgroundFunctionally coupled large myocardial grafts and a remarkable improvement of heart function in nonhuman primate models of myocardial infarction have been reported after transplantation of human embryonic stem cell-derived cardiomyocytes at relatively high numbers of up to 109 single cell cardiomyocytes - a dose equivalent to total cell loss after myocardial infarction in [~]10 times larger human hearts. To overcome apparent limitations associated with the application of single cells, this pre-clinical study investigated the injection of cardiomyocyte aggregates instead. MethodsHuman iPSC-derived cardiomyocyte aggregates were produced in scalable suspension culture. Intramyocardial injection of the aggregates into cynomolgus monkey hearts was conducted two weeks after myocardial infarction induced by permanent coronary artery ligation. Human cell engraftment was assessed after two weeks or three months; functional analyses included continuous telemetric ECG recording and repeated cardiac MRI assessment in comparison to sham treated animals. ResultsTreatment with cell numbers as low as 5 x 107 resulted in efficient structural engraftment. Notably, the degree of heart function recovery in vivo seemed to correlate with the contractility of the applied cardiomyocytes tested by parallel experiments in vitro. Graft-induced non-life-threatening arrhythmias were transient and decreased considerably during the three months follow-up. ConclusionsTransplantation of human iPSC-derived cardiomyocyte aggregates yielded comparable results to the reported application of higher numbers of single cell cardiomyocytes from human ESC, suggesting that the application of cardiomyocyte aggregates facilitates cell therapy development by reducing cell production costs and clinical risks associated with the administration of relatively high cell numbers. Clinical PerspectiveWhat is new? O_LIIn contrast to previously applied single cells, human iPSC-derived cardiomyocyte aggregates (hiCMAs) were transplanted in a non-human primate (NHP) model of MI, to reduce the required cell dose, promote myocardial retention of the graft, and limit the risks for adverse effects. Such low-dose treatment with almost pure ventricular cardiomyocytes produced under GMP-compliant conditions, resulted in the formation of relative large, structurally integrated human grafts in NHP hearts. C_LIO_LITransient non-life-threatening arrhythmias associated with intramyocardial cell transplantation decreased considerably during the three months follow-up. C_LIO_LIA remarkable recovery of left ventricular function was observed. This recovery notably correlated with the in vitro contractility of transplanted cardiomyocyte batches tested in bioartificial cardiac tissues (BCTs), underlining the relevance of a suitable potency assay. C_LI What are the clinical implications? O_LIIntra-myocardial injection of hiCMAs is a promising treatment modality for the recovery of contractile function after MI; their advanced production, storage and testing revealed in the study facilitate the clinical translation of hiPSC-based heart repair. C_LIO_LIThe need for relatively low numbers of cardiomyocytes produced through advanced protocols for scalable suspension culture reduces production costs of adequate cell batches, thereby increasing treatment availability. In vitro testing of the produced cell batches is required to ensure treatment efficacy. C_LIO_LIClinical hiCMA injection can be considered reasonably safe, however, pharmacological prevention and treatment of arrhythmias is required and temporary implantation of a cardioverter-defibrillator (ICD) could be considered. C_LI

During cell culture, trypsin, a serine protease, is applied to cells for 5-10 minutes to separate them from each other and from the underlying substratum so that they can be transferred to a different vessel, for re-plating, after growth medium containing 10 % serum has been added to the cells, in a well-known technique known as passaging. The serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of trypsin, elastase and other serine proteases. Although what is used is bovine serum in which levels of proteins could be different from levels seen in humans, normal human serum contains A1AT (> 1 mg/ml; > [~]18 {micro}mol/L) as well as trypsin itself (< 460 ng/ml, or [~]0.02 {micro}mol/L), with the former in a [~]900-fold molar excess over the latter. Thus, it may be assumed there is also enough A1AT in the bovine serum added during passaging, to neutralize the trypsin ([~]100 M) present in the small volume of trypsin-EDTA solution used to separate cells. What are the consequences of not adding serum, when growth medium is added, or of maintaining cells for a few tens of hours in the presence of trypsin, in a serum-free growth medium? What does such sustained exposure to trypsin during cell culture do to cells? More generally, what are the responses of cells within an organism to the balance of trypsin and A1AT in the serum that bathes them constantly? We know that excesses and deficiencies in the levels of either trypsin or A1AT are associated with disease. We know that cellular metabolism can be influenced through signaling involving protease activated membrane GPCR receptors (PAR1-4). In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. We also know that cells at sites of injury or inflammation produce and secrete trypsin, and that this trypsin can act locally upon cells in a variety of ways, all of which have probably not yet been elucidated. Here, we show that sustained exposure to trypsin induces cells to de-differentiate into a stem-like state. We show that if serum is either not added at all, or added and then washed away (after confluency is attained), during cell culture, all cells exposed to exogenously-added trypsin undergo changes in morphology, transcriptome, secretome, and developmental potential, and transition into a state of stemness, in minimal essential medium (MEM). Regardless of their origins, i.e., independent of whether they are derived from primary cultures, cell lines or cancer cell lines, and regardless of the original cell type used, exposure to trypsin ([~]10 {micro}M; [~]250 {micro}g/ml) at a concentration 10-fold lower than that used to separate cells during passaging ([~]100 M), over a period of 24-48 hours, causes cells to (1) become rounded, (2) cluster together, (3) get arrested in the G0/G1 stage of the cell cycle, (4) display increased presence of 5-hydroxymethyl cytosine in their nuclei (indicative of reprogramming), (5) display increased levels of activated PAR2 membrane receptor, (6) become capable of very efficient efflux of drug-mimicking dyes, (7) express factors and/or markers known to be associated with induction and/or attainment of stemness, with predominant expression of Sox-2 within cell nuclei; (8) display overall transcriptomic (RNASEQ) profiles characteristic of stemness; (9) secrete stemness-associated factors such as bFGF, and IL-1{beta}, into the medium, in quantities sufficient to support autocrine function (in certain cases); and (10) display increased conversion of pro-MMPs into activated MMPs in the cells secretome. Notably, (11) inclusion of differentiating and/or transdifferentiating factors in the environment of such cells causes them to express markers associated with ectodermal, endodermal and mesodermal cell lineages and/or transdifferentiate into specific cell types, e.g., adipocytes or osteocytes.\n\nMost intriguingly of all, (12) the attained stemness appears to be reversible, i.e., withdrawal of trypsin from the medium prior to addition of any differentiating factors restores cells to their original morphology, also over a period of 24-48 hours. Further, (13) a known PAR2 agonist, and a known PAR2 antagonist, respectively, appear to mimic effects of trypsin addition and withdrawal/inhibition. In addition, (14) in experiments with a particular cancer characterized by high levels of stemness (TNBC; triple negative breast cancer), tissues of all TNBC patients express high levels of the PAR2 receptor, as do cells from a known TNBC-derived cell line. We propose that through their effects on PAR levels, and PAR activation status, the balance of trypsin and A1AT levels in organisms normally regulates cellular potential for differentiation, de-differentiation or transdifferentiation, in a local manner, with the default status being that A1AT inhibits trypsin and keeps cells differentiated, whereas sustained trypsin signaling at sites of injury through local production of trypsin helps to place cells into an intermediate state of stemness from which they can either return to their original differentiated state(s), or undergo factor-dependent differentiation, or transdifferentiation, into specific cell types or lineages. It is also possible that reduction in A1AT promotes regeneration. We present a core (RNASEQ-derived) signature for trypsin-induced stemness in human corneal fibroblasts (HCFs) and cells from a retinal pigment epithelial cell line (ARPE-19), noting that there are commonalities as well as differences between them, which suggests that this core signature will be amended with RNASEQ studies of more trypsin-exposed cell types. Our findings offer a possible explanation for the recent unexplained increase in the preference for serum-free protocols used for induction and maintenance of stemness involving iPSCs and mesenchymal stem cells. Also, our studies suggest a new approach to understanding and exploiting how organisms might use stemness, in adults. Trypsin-dominated serine protease induced reprogramming (SPIR) might offer a more natural, and suitably softer, method of reprogramming of cellular developmental potential for local regenerative requirements in animal tissues.

RationaleCold static preservation at 4{degrees}C is the clinical standard for donor lung storage but is limited to 6-8 hours of cold ischemia. Static storage at 10{degrees}C has been shown to extend ischemia times and improve lung health. Given that lungs can maintain aerobic metabolism ex vivo, we hypothesized that adding ventilation at 10{degrees}C would further prolong preservation by stimulating aerobic metabolism. MethodsLungs were procured from C57Bl/6 mice and then stored for 24h with ventilation at 10{degrees}C (n=4), statically at 10{degrees}C (n=4), or statically at 4{degrees}C (n=4). Respiratory mechanics were evaluated using a FlexiVent system. Cellular viability was assessed via flow cytometry. Complement shedding was evaluated by enzyme-linked immunosorbent assay. Histologic evidence of lung injury was assessed by H&E staining. ResultsDonor lungs stored with ventilation at 10{degrees}C exhibited significantly reduced histologic injury scores compared to static storage at 4{degrees}C (p = 0.0062). Ventilation also decreased complement C3 shedding (p < 0.01), apoptosis (p < 0.05), cytochrome c release (p = 0.0014), and ROS production (p = 0.0008) compared to statically stored lungs at 4{degrees}C and 10{degrees}C. Functionally, ventilated lungs demonstrated improved respiratory mechanics with lower airway resistance (p = 0.021) and increased compliance (p = 0.023) compared to static storage at 10{degrees}C. ConclusionsVentilating lungs at 10{degrees}C compared to static cold storage appears to result in healthier and more functional lung tissue and may extend the preservation times of donor organs for lung transplantation.