Main Text Just years since the development of
Just 10 years since the development of human induced pluripotent stem cell (hiPSC) technology (Takahashi et al., 2007), the use of these cells to model lpl receptor disorders and obtain disease-relevant information is becoming a tangible reality. Not only are we now able to better detect relevant genetic changes in a patient’s cells using high-throughput genome sequencing technology but also we can establish a direct phenotypic correlation between genetic mutations and an aberrant neuronal phenotype or developmental trajectory. The latest improvements in generating relevant neural cell types by either differentiation of hiPSC lines or by direct conversion of somatic cells (e.g., fibroblasts) now allow researchers to make cells from different areas of the central nervous system (CNS) and peripheral nervous system (PNS) and probe effects on the cell type where disease manifests. This represents a significant improvement of previous experimental tools, including animal models and in vitro cultures of non-relevant cell lines (such as 293T or HeLa cells), which recapitulate only some of the specific traits of human disease (Eglen and Reisine, 2011; Pouton and Haynes, 2005), with the potential to reverse the current trend of huge investments by the pharmaceutical industry yielding few therapeutic compounds entering the market (Mullard, 2015; Scannell et al., 2012).
In April 2015, a group of stem cell researchers, neuroscientists, genomic and computational biologists, clinicians, and industry partners met for 4 days at the Banbury Center at Cold Spring Harbor, New York, to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders (Figures 1 and 2). This opinion piece outlines the current state of the field and discusses the main challenges that should drive future research initiatives.
Introduction Monozygotic (MZ) twins originate from the same conception event and are genetically identical. Approximately 80% of MZ twins originate from monochorionic/diamniotic pregnancies, meaning that they share the placenta and chorion, whereas each twin has its own amnion. These monochorionic/diamniotic MZ twins form if the inner cell mass (ICM), the part of the embryo that will give rise to the fetal body, splits at the early blastocyst stage, 4–6 days post-fertilization. Molecular events leading to this splitting are still in the domain of speculation. Time-lapse cinematography suggests that, at least in vitro, the reason might be purely mechanical; twins have been shown to form after blastocyst collapse and re-expansion, splitting the ICM into two groups of cells (Payne et al., 2007). It is well documented that the incidence of MZ twins is higher following assisted reproduction techniques (Blickstein et al., 2003; Vitthala et al., 2009; Knopman et al., 2014; Tocino et al., 2015); however, we have rarely seen a good-quality blastocyst with two distinct ICMs among embryos donated for research in our center. High-resolution single-cell transcriptome analysis of the temporal and spatial patterns of gene expression in the human preimplantation embryos has been reported (Xue et al., 2013; Yan et al., 2013), and when we recently encountered such an embryo, we took the opportunity to compare the ICMs at the transcriptome level using next-generation sequencing (NGS).
Results The expanded day 6 blastocyst displayed two distinct ICMs 4 hr post-thawing, (Figure 1A). Single cells and cell borders were clearly visible in ICM1, whereas the cells in ICM2 were more tightly packed and no single cell could be distinguished, indicating that ICM2 is either better quality or more advanced than ICM1. To further explore this morphological discrepancy, we separated ICM1 and ICM2 from trophectoderm (TE) and by NGS evaluated the transcriptome of each of the three fractions. Tiered quality-control (QC) steps suggested good quality of cDNA and the library (Figure S1), as well as high homogeneity between samples (Figure S2). Nevertheless, we are aware that comparing two conditions with no replicates is not ideal and strong events and false positives may be missed.