Stem Cell Dynamics Research Articles

Transcriptional Heterogeneity of Neural Stem Cells during Axolotl Salamander Spinal Cord Regeneration

Unlike humans, axolotl salamanders are capable of restoring sensory and motor function after spinal cord injury. Upon tail amputation or spinal cord transection, endogenous neural stem cells (NSCs) increase proliferation and differentiate into multiple neural and glial cell types of the central and peripheral nervous system. How NSCs transition from a resting state to a regenerative state and coordinate proper neural differentiation is poorly understood. Here, we study the global changes that occur in NSCs after spinal cord injury as well as specific changes in gene transcription as the NSC regenerative program unfolds. We first studied global changes in NSCs after injury by measuring global RNA synthesis temporally and spatially through incorporation of 5‐ethynyl‐uridine into nascent RNA. Substantial upward shifts in overall transcriptional output occurred in specific NSC populations during regeneration, which was correlated with loss of facultative repressive chromatin marks, and was dependent on Hippo‐YAP signaling. We also found an evolutionarily conserved upregulation of nascent RNA production in active neural progenitors in axolotls and mice. In order to dig deeper into neural stem cell dynamics during regeneration, we adopted multiplexed fluorescence in situ hybridization (FISH) in tissue section and whole mount tissues. We produced a user‐friendly, sharable, Docker Container for automated high‐throughput design of Third Generation Hybridization Chain Reaction (HCR) FISH probe sets. An advantage of HCR‐FISH is that it utilizes non‐enzymatic signal amplification, while maintaining low background signal, leading to high signal‐to‐noise detection of mRNAs. We demonstrate that the HCR‐FISH method is amenable to multiplexing through five sequential rounds of hybridization (SeqFISH). Using our probe design pipeline and HCR‐FISH, we imaged NSC activation and differentiation in tissue sections collected after injury using 15 simultaneously‐detected probe sets indicative to CNS and PNS cell types. Whole mount HCR‐FISH for three neural markers were also imaged simultaneously to assess NSC and neuron responses after injury. Overall, the genetic neuroanatomical mapping performed here using molecular classification of NSCs provides novel information on how NSCs respond to injury to repair a CNS injury.Support or Funding InformationThis project was funded by grants NSF 1656429, NSF 1558017, and NIH OD024909

MCL-1 Inhibition by Selective BH3 Mimetics Disrupts Mitochondrial Dynamics Causing Loss of Viability and Functionality of Human Cardiomyocytes.

SummaryMCL-1 is a well-characterized inhibitor of cell death that has also been shown to be a regulator of mitochondrial dynamics in human pluripotent stem cells. We used cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) to uncover whether MCL-1 is crucial for cardiac function and survival. Inhibition of MCL-1 by BH3 mimetics resulted in the disruption of mitochondrial morphology and dynamics as well as disorganization of the actin cytoskeleton. Interfering with MCL-1 function affects the homeostatic proximity of DRP-1 and MCL-1 at the outer mitochondrial membrane, resulting in decreased functionality of hiPSC-CMs. Cardiomyocytes display abnormal cardiac performance even after caspase inhibition, supporting a nonapoptotic activity of MCL-1 in hiPSC-CMs. BH3 mimetics targeting MCL-1 are promising anti-tumor therapeutics. Progression toward using BCL-2 family inhibitors, especially targeting MCL-1, depends on understanding its canonical function not only in preventing apoptosis but also in the maintenance of mitochondrial dynamics and function.

Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation.

Stem Cell Dynamics and Cytoceuticals for Therapeutics and Toxicity Screening: A Review

Cellular therapy involves use of whole cell or cell cytoplasmic macro-molecular inclusions and their derivatives as resources for treatment and management of diseases and their complications in animals and humans. The use of these biological and bio-medical resources in modern times is increasingly gaining popularity within the community of health care givers.

Isolation of satellite cells and transplantation into mice for lineage tracing in muscle.

Limited methods exist to assay the direct effects of therapeutic intervention on muscle stem cell fate, proliferation or differentiation in an in vivo context. Here we provide an optimized protocol for muscle stem cell isolation and transplantation into mice to deconvolute heterogeneity within isolated stem cell populations. Viable and pure cell populations are isolated within 2 h and can then be used for therapeutic intervention or transplantation to uncover the repopulating and differentiation potential in mice, a physiologically relevant in vivo context. Effects can be assessed 9 d after transplantation. This methodology analyzes cell and sort purity prior to transplantation to improve reproducibility and outlines novel blocking steps to improve tissue staining and analysis. Experience with surgical procedures in mice is recommended before attempting this protocol. Our system is widely applicable for exploring stem cell dynamics within muscle and has already been used to study heterogeneity within muscle stem cell populations and efficacy of therapeutic intervention on isolated stem cell populations.

Dynamics of cardiomyocyte and muscle stem cell proliferation in pig

The cardiac and skeletal muscle tissues are both striated and contractile but their intrinsic cellular properties are distinct. The minimal cardiomyocyte proliferation and the lack of cardiac stem cells directly leads to poor heart repair in adult mammals. But in skeletal muscle, the robust proliferation of widespread muscle stem cells support efficient muscle regeneration. The endogenous cardiomyocyte and muscle stem cell proliferation has been analyzed in common laboratory animals but not in large mammals including pigs, which are more comparable to human. In this study, we rigorously examined the cell cycle dynamics of porcine cardiomyocytes and muscle stem cells through different developmental stages. Proliferative cardiomyocytes and muscle stem cells were broadly observed in the embryonic heart and limb muscle respectively. Muscle stem cells continue to proliferate postnatally but cardiomyocyte proliferation was drastically reduced after birth. However, robust cardiomyocyte cell cycle activity was detected around postnatal day 20, which could be attributed to the binucleation but not cell division. Increased proliferating cells were detected in maternal heart during early pregnancy but they represent non-cardiomyocyte cell types. The islet1 expressing cells were only identified in the embryonic and new born porcine hearts. Furthermore, the accumulated oxidative DNA damage in the cardiac but not skeletal muscle during development could be responsible for the diminished cardiomyocyte proliferation in adult pig. Similarities and differences in the proliferation of heart and skeletal muscle cells are identified in pigs across different developmental stages. Such cellular proliferative features must be taken into account when using porcine models for cardiovascular and muscular research.

P036 A novel p53-mediated mouse model of inflammatory bowel disease-associated colorectal cancer

Abstract Background Current mouse models of colitis-associated cancer often utilise a combination of APC mutation, Dextran sulphate sodium (DSS) and azoxymethane administration. However, only 13% of human inflammatory bowel disease (IBD)-associated cancers is associated with an APC mutation1. Instead, they are frequently associated with mutation of p53 (occurring in 63% of human cancers1). Whilst some models of IBD-associated cancer using either global p53 loss or mutant p53 have been described, we felt that a more pathophysiologically relevant model would be represented using p53 loss solely in the colonic epithelium. Methods Colonic epithelial p53 floxing in Vil-CreERT2p53fl/fl mice was achieved through intraperitoneal injection of 120 and 80 mg/kg tamoxifen on two consecutive days. After 16 days, mice were started on a chronic DSS treatment protocol. Chronic DSS-induced colitis involved three 5-day cycles of low-dose DSS (1.5% initially and subsequently 1% for the remaining two cycles), interspersed with normal drinking water for 2 weeks to facilitate recovery. After three cycles of DSS, mice were aged to 164 days. Results At 164 days post induction of DSS, 100% of mice had developed adenomata with low-grade dysplasia (n = 4). Adenomata in 50% of these mice (n = 2) also had features of high-grade dysplasia. There were between one and three adenomata in each animal. No features of invasive adenocarcinoma were demonstrated. There was partial β-catenin nuclear localisation in these tumours. Background chronic colitis was evident, even though the experiment ended over 3 months after the DSS treatment finished. Conclusion To the best our knowledge, this is the first mouse model of colitis-associated cancer using p53 loss in the colonic epithelium together with chronic colitis induction using DSS. The experiment ended over 3 months after the DSS treatment finished, showing that the resultant chronic colitis can persist long after the initial insult is given. The fact that β-catenin is partially, but not fully nuclear localised suggests an alternative pathway to tumour formation in this p53fl/fl model, and may therefore be more pathophysiologically relevant to human IBD-associated colorectal cancers. Further work is now being carried out to both validate and further evaluate this novel mouse model, including assessment of stem cell dynamics in response to colitis in the context of p53 loss. Reference

Mouse Model for Sporadic Mutation of Target Alleles to Understand Tumor Initiation and Progression and Stem Cell Dynamics.

Recent evidence has shown that many different tissues accumulate mutations even though the tissue is phenotypically normal. Therefore, generating mouse models for visualizing the tissue level effects that happen after oncogenic mutation in a single, isolated cell are critical for understanding tumor initiation and the role of competition in stem cell dynamics. Most mouse models have oncogenic mutations at the level of the entire mouse, the entire tissue, or all cells of a specific type in a tissue. However, these mouse models do not mimic the microenvironmental interactions that occur after an isolated cell acquires an oncogenic mutation because of the large number of mutant cells. We developed a mouse model for sporadic and isolated mutation of target alleles to better address the questions of sporadic cancer and stem cell competition. The following chapter describes methods for utilizing this mouse model and a few examples of the novel findings of using such a model.

Aging induces aberrant state transition kinetics in murine muscle stem cells.

ABSTRACTMurine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.

Dynamics of competing heterogeneous clones in blood cancers explains multiple observations - a mathematical modeling approach.

Heterogeneity of stem cell clones provide a key ingredient in altered hematopoiesis and is of main interest in the study of predisease states as well as in the development of blood cancers such as chronic myeloid leukemia (CML) and the Philadelphia-negative myeloprofilerative neoplasms (MPNs). A mathematical model based on biological mechanisms and basic cell descriptors such as proliferation rates and apoptosis rates is suggested, connecting stem cell dynamics with mature blood cells and immune mediated feedback. The flexible approach allows for arbitrary numbers of mutated stem cell clones with perturbed properties. In particular, the stem cell niche provides a competition between wild type and mutated stem cells. Hence, the stem cell niche can mediate suppression of the wild type clones and up-regulation of one or more malignant clones. The model is parameterized using clinical data to show typical disease progression in several blood cancers and the hematological and molecular response to treatment. Intriguingly, occasional oscillatory cell counts observed during treatment of CML and MPNs can be explained by heterogeneous stem cell clone dynamics. Thus, the vital heterogeneous stem cell dynamics may be inferred from mathematical modeling in synergy with clinical data to elucidate hematopoiesis, blood cancers and the outcome of interventions.

Organoid Derivation and Orthotopic Xenotransplantation for Studying Human Intestinal Stem Cell Dynamics.

Intestinal stem cells continuously self-renew throughout life to maintain gut homeostasis. With the advent of the organoid culture system, we are now able to indefinitely expand healthy and diseased tissue-derived human intestinal stem cells in vitro and use them for various applications. Nonetheless, investigating the behavior of human intestinal stem cells in vivo still remains challenging. We recently developed an orthotopic xenotransplantation system that realizes in vivo reconstruction of human intestinal epithelial tissue with preserved stem cell hierarchy by engrafting human normal colon organoids onto the mouse colon surface. We also introduced new growth factors, namely, insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2), into the culture condition for human intestinal organoids that significantly increase scalability and transfectability of the organoids. By integrating these recent advances, we organized a tissue-oriented platform encompassing derivation of patient-derived intestinal organoids and their orthotopic xenotransplantation. The research platform based on orthotopic xenotransplantation of human intestinal organoids provides a powerful tool for studying human intestinal stem cell biology in native tissue-relevant contexts as well as for establishing novel disease modeling systems.

Chromatin regulation and dynamics in stem cells.

In eukaryotes, DNA is highly compacted within the nucleus into a structure known as chromatin. Modulation of chromatin structure allows for precise regulation of gene expression, and thereby controls cell fate decisions. Specific chromatin organization is established and preserved by numerous factors to generate desired cellular outcomes. In embryonic stem (ES) cells, chromatin is precisely regulated to preserve their two defining characteristics: self-renewal and pluripotent state. This action is accomplished by a litany of nucleosome remodelers, histone variants, epigenetic marks, and other chromatin regulatory factors. These highly dynamic regulatory factors come together to precisely define a chromatin state that is conducive to ES cell maintenance and development, where dysregulation threatens the survival and fitness of the developing organism.

Histone H3 K4/9/27 Trimethylation Levels Affect Wound Healing and Stem Cell Dynamics in Adult Skin.

SummaryEpigenetic mechanisms controlling adult mammalian stem cell (SC) dynamics might be critical for tissue regeneration but are poorly understood. Mouse skin and hair follicle SCs (HFSCs) display reduced histone H3 K4me3, K9me3, and K27me3 methylation levels (hypomethylation) preceding hair growth. Chemical inhibition of relevant histone demethylases impairs subsequent differentiation and growth of HFs and delays wound healing. In wounding, this impairs epithelial cell differentiation and blood vessel recruitment, but not proliferation and fibroblast recruitment. With Aspm-CreER as a newfound inter-follicular epidermis lineage-labeling tool, and Lgr5-CreER for hair follicles, we demonstrate a reduced contribution of both lineages to wound healing after interfering with hypomethylation. Blocked hypomethylation increases BMP4 expression and selectively upregulates H3 K4me3 on the Bmp4 promoter, which may explain the effects on HFSC quiescence, hair cycle, and injury repair. Thus, transient hypomethylation of histone H3 K4/9/27me3 is essential for adult skin epithelial SC dynamics for proper tissue homeostasis and repair.

Bacterial Shoot Apical Meristem Inoculation Assay.

By virtue of their sessile nature, plants may not show the fight-and-flight response, but they are not devoid of protecting themselves from disease-causing agents, attack by herbivores, and damages that are caused by other environmental factors. Plants differentially protect their life-sustaining organs such as plant apexes from the attack by microbial pathogens. There are well-established methods to inoculate/infect various plant parts such as leaves, roots, and stems with various different pathogens. The plant shoot apical meristems (SAM) are a high-value plant target that provides niche to stem cell populations. These stem cells are instrumental in maintaining future plant progenies by giving birth to cells that culminate in flowers, leaves, and stems. There are hardly few protocols available that allow us to study immune dynamics of the plant stem cells as they are hindered by various layers of the SAM cell populations. Here, we describe a step-by-step method on how to inoculate the Arabidopsis SAM with model plant pathogen Pseudomonas syringae pv. tomato DC3000.

Efficient depletion of ribosomal RNA for RNA sequencing in planarians

BackgroundThe astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.ResultsIn this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium.ConclusionsThe ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

Capturing Stem Cell Behavior Using Intravital and Live Cell Microscopy.

Stem cells maintain tissue homeostasis by driving cellular turnover and regeneration upon damage. They reside within specialized niches that provide the signals required for stem cell maintenance. Stem cells have been identified in many tissues and cancer types, but their behavior within the niche and their reaction to microenvironmental signals were inferred from limited static observations. Recent advances in live imaging techniques, such as live cell imaging and intravital microscopy, have allowed the visualization of stem cell behavior and dynamics over time in their (near) native environment. Through these recent technological advances, it is now evident that stem cells are much more dynamic than previously anticipated, resulting in a model in which stemness is a state that can be gained or lost over time. In this review, we will highlight how live imaging and intravital microscopy have unraveled previously unanticipated stem cell dynamics and plasticity during development, homeostasis, regeneration, and tumor formation.

Identification of an Expanded Immature Cell Population in Human Bone Marrow during Erythroid Responses to ESA Treatment

Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment. Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR. Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression (<0.5% of CD34+CD38- compartment; median 0.15%; range 0.04 - 0.5%; n = 5) in normal BM samples and a similar frequency in MDS-RS with stable anemia (median 0.17%; range <0.01 - 1.12%; n = 9). However, in MDS-RS BM samples taken during ESA response, the CD34+CD38-CD105+ population increased up to 13 fold (range 2 - 13; n = 4). Interestingly, this population returned to the low baseline level, similar to normal BM, when the patients became refractory to ESA. While SF3B1 mutation burden in total BM mononuclear cells was not correlated with degree of anemia or treatment need, VAFs in CD34+ cells were significantly lower in patients with stable anemia compared to those with treatment need (p < 0.01). A similar pattern was observed in the mature CD34+CD38+ populations CMP, GMP and MEP (p < 0.05). Interestingly, in patients responding to ESA treatment, the erythroid-committed MEPCD105+ population was generally completely mutated, while the mutation burden in the expanding aberrant CD34+CD38-CD105+ population was much lower in several patients. Intriguingly, aberrant expression of CD105 was also found in mature erythroblast populations from ESA-responding MDS patients. Conclusion: Our data indicate that presence of wildtype CD34+ BM populations in anemic MDS-RS patients is associated with better erythroid output and milder anemia. Our results show poor discriminative power of SF3B1 mutational burden in total BM as routinely assessed in the clinic, whereas SF3B1 VAF in defined CD34+ HSPC populations shows a strong correlation with treatment need. Expression of the surface marker CD105 identifies an immature CD34+CD38- BM population that expands during erythroid responses to ESA treatment. Because this population also can be detected at low levels in normal BM donors, and has a lower mutation burden than other HSPC populations in MDS-RS patients, we propose that this wildtype population acts as a reserve source of erythroblasts during stress erythropoiesis, possibly bypassing the traditional hierarchy. Disclosures No relevant conflicts of interest to declare.

Symposium review: Determinants of milk production: Understanding population dynamics in the bovine mammary epithelium

The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.

MCL-1 Inhibition by Selective BH3 Mimetics Disrupts Mitochondrial Dynamics in iPSC-Derived Cardiomyocytes

MCL-1 is a well characterized inhibitor of cell death that has also been shown to be a regulator of mitochondrial dynamics in human pluripotent stem cells. We used cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to uncover whether MCL-1 is crucial for cardiac function and survival. Inhibition of MCL-1 by BH3 mimetics resulted in the disruption of mitochondrial morphology and dynamics as well as disorganization of the actin cytoskeleton. Interfering with MCL-1 function affects the homeostatic proximity of DRP-1 and MCL-1 at the outer mitochondrial membrane, resulting in decreased functionality of hPSC-CMs. Cardiomyocytes display abnormal functional cardiac performance even after caspase inhibition, supporting a non-apoptotic activity of MCL-1 in hiPSC-CMs. BH3 mimetics targeting MCL-1 are promising anti-tumor therapeutics. Progression towards using BCL-2 family inhibitors, especially targeting MCL-1, depends on understanding not only its canonical function in preventing apoptosis, but also in the maintenance of mitochondrial dynamics and function.

Applications of Computer Modeling and Simulation in Cartilage Tissue Engineering.

Advances in cartilage tissue engineering have demonstrated noteworthy potential for developing cartilage for implantation onto sites impacted by joint degeneration and injury. To supplement resource-intensive in vivo and in vitro studies required for cartilage tissue engineering, computational models and simulations can assist in enhancing experimental design. Research articles pertinent to cartilage tissue engineering and computer modeling were identified, reviewed, and summarized. Various applications of computer modeling for cartilage tissue engineering are highlighted, limitations of in silico modeling are addressed, and suggestions for future work are enumerated. Computational modeling can help better characterize shear stresses generated by bioreactor fluid flow, refine scaffold geometry, customize the mechanical properties of engineered cartilage tissue, and model rates of cell growth and dynamics. Thus, results from in silico studies can help resourcefully enhance in vitro and in vivo studies; however, the limitations of these studies, such as the underlying assumptions and simplifications applied in each model, should always be addressed and justified where applicable. In silico models should also seek validation and verification when possible. Future studies may adopt similar approaches to supplement in vitro trials and further investigate effects of mechanical stimulation on chondrocyte and stem cell dynamics. Additionally, as precision medicine, machine learning, and powerful open-source software become more popular and accessible, applications of multi-scale and multiphysics computational models in cartilage tissue engineering are expected to increase.