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The rapidly growing field of cellular engineering is enabling scientists to more effectively create in vitro models of disease and develop specific cell types that can be used to repair damaged tissue. In particular, the engineering of neurons and other components of the nervous system is at the forefront of this field. The methods used to engineer neural cells can be largely divided into systems that undergo directed differentiation through exogenous stimulation (i.e., via small molecules, arguably following developmental pathways) and those that undergo induced differentiation via protein overexpression (i.e., genetically induced and activated; arguably bypassing developmental pathways). Here, we highlight the differences between directed differentiation and induced differentiation strategies, how they can complement one another to generate specific cell phenotypes, and impacts of each strategy on downstream applications. Continued research in this nascent field will lead to the development of improved models of neurological circuits and novel treatments for those living with neurological injury and disease.

Chemotherapy‐induced peripheral neuropathy (CIPN) is a dose‐limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell–derived sensory neuron (iPSC‐SN) model was developed for the study of chemotherapy‐induced neurotoxicity. The iPSC‐SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca2+ influx in response to capsaicin, α,β‐meATP, and glutamate. The iPSC‐SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half‐maximal inhibitory concentration (IC50) values of 38.1 µM (95% confidence interval (CI) 22.9–70.9 µM) for 48‐hour exposure and 9.3 µM (95% CI 5.7–16.5 µM) for 72‐hour treatment. Paclitaxel causes dose‐dependent and time‐dependent changes in neurite network complexity detected by βIII‐tubulin staining and high content imaging. The IC50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3–16.9 µM) for 48‐hour exposure and 0.6 µM (95% CI 0.09–9.9 µM) for 72‐hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate‐induced neuronal excitability were also observed with paclitaxel exposure. The iPSC‐SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC‐SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy‐induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment.

Summary: The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and limitations in robust in vitro derivation methods. We developed a dual SHOX2:GFP; MYH6:mCherry knockin human embryonic stem cell (hESC) reporter line, which allows the identification and purification of SAN-like cells. Using this line, we performed several rounds of chemical screens and developed an efficient strategy to generate and purify hESC-derived SAN-like cells (hESC-SAN). The derived hESC-SAN cells display molecular and electrophysiological characteristics of bona fide nodal cells, which allowed exploration of their transcriptional profile at single-cell level. In sum, our dual reporter system facilitated an effective strategy for deriving human SAN-like cells, which can potentially be used for future disease modeling and drug discovery.

5-fluorouracil (5-FU) is among the mostly administrated chemotherapeutic agents for a wide variety of neoplasms. Non-coding RNAs have a central impact on the determination of the response of patients to 5-FU. These transcripts via modulation of cancer-related pathways, cell apoptosis, autophagy, epithelial–mesenchymal transition, and other aspects of cell behavior can affect cell response to 5-FU. Modulation of expression levels of microRNAs or long non-coding RNAs may be a suitable approach to sensitize tumor cells to 5-FU treatment via modulating multiple biological signaling pathways such as Hippo/YAP, Wnt/β-catenin, Hedgehog, NF-kB, and Notch cascades. Moreover, there is an increasing interest in targeting these transcripts in various kinds of cancers that are treated by 5-FU. In the present article, we provide a review of the function of non-coding transcripts in the modulation of response of neoplastic cells to 5-FU.

The early molecular events that ultimately lead to neuronal cell death in pathologies such as Parkinson’s disease are poorly understood. Here the authors use pluripotent stem-cell-derived human midbrain neurons and chemical biology tools to gain molecular level insight into the events induced by toxic and genetic stresses that mimic those occurring during neurodegeneration.

Summary: The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identified ALCAM (CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications. : In this article, Salekdeh and colleagues show that ISL1+ cardiac progenitors can be purified from a heterogeneous population of hESC-derived cardiomyocytes using ALCAM. Transplantation of multipotent ISL1+/ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis in a rat model of myocardial infarction, based on cardiac MRI and histology. Keywords: cell therapy, proteomics, myocardial biology, stem cells

BACKGROUND: Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. METHODS: We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells. RESULTS: We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line. CONCLUSIONS: This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation.