Defining and Controlling the Subtype Identity of Human Stem Cell-Derived Motor Neurons

One cardinal promise of stem cell research is that many intractable, common, and poorly understood diseases may be studied in an entirely new way: in vitro in the specific human cell types affected in vivo. Embryonic stem (ES) cells have the pluripotency to generate all somatic cells types, and the invention of somatic cell reprogramming techniques has allowed the creation of cell lines with both ES-cell grade pluripotency--induced pluripotent stem (iPS) cells--and the genetic determinants of diseases. If iPS cells derived from patients with genetic disease are to enable studying the affected human cell types in vitro then it is necessary to: first, precisely define the appropriate cellular phenotypes in vivo; second, selectively generate those cell types in vitro; and third, demonstrate that iPS cells retain similarly predictable and tractable cellular potential as ES cells. In the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS) spinal motor neurons innervating different types of muscles and individual muscle groups show selective vulnerability or resistance to disease. We therefore set out to define the subtypes of human motor neurons in vivo and to generate these in vitro. Here we report that human motor neurons in vivo share with mouse the molecular markers of motor neuron column, division, and pool organization, as well as positional expression of HOX proteins which regulate this diversity in chick and mouse. We then used combinations of these markers to classify motor neuron subtypes derived from human ES cells in vitro under standard differentiation conditions. These human ES cell-derived motor neurons expressed marker combinations appropriate to each motor column, but were strongly biased to cervical phenotypes. In order to access a greater diversity of motor neuron subtypes, including some with differential responses to ALS in vivo, we defined a developmental strategy to generate more caudal ES-cell derived motor neurons. We show that FGF treatment, in a patterning window we defined, generated human ES-cell derived motor neurons with more caudal (brachial, thoracic, and lumbar) phenotypes. We then participated in a long term collaboration to generate iPS cell lines from donors with ALS-genotypes (familial ALS), and no clinical motor dysfunction (controls). We first showed that ALS and control iPS cells from patients of advanced age could generate motor neurons in vitro. To address questions about the variability of iPS cells, and their comparability to ES cells for making defined neuronal subtypes, we generated a panel of iPS lines from donors of varying demography, thoroughly characterized these cells by standard assays for pluripotent cells, and assessed their ability to generate functional motor neurons in comparison to a panel of ES cell lines. We showed that iPS cells were equivalent to ES cells, and that human genetic diversity may influence the efficiency of motor neuron generation. Next, we used these lines to show that iPS cells could generate the same diversity of motor neurons in vitro, and that the rostrocaudal output of this diversity was rationally manipulable. Finally, since ALS is an adult onset disease, we anticipated that if ES and iPS cell-derived motor neurons could reach significant landmarks of functional maturation in vitro, then the chances of manifesting disease phenotypes would be increased. Therefore we developed methods for long term cultures in which ES and iPS cell-derived motor neurons showed progressive molecular, morphological, and electrophysiological maturation. Together these results enable future studies to ask if ALS-patient iPS cell-derived motor neurons will show pan-motor neuron or subtype-specific ALS phenotypes in vitro. In turn these which may help elucidate mechanisms of disease resistance and vulnerability and identify novel therapeutic targets

Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases

A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use

Abstract Background It has become increasingly apparent that the trophectoderm (TE) at blastocyst stage is much more mosaic than has been appreciated. Whether preimplantation genetic screening (PGS), utilizing a single TE biopsy (TEB), can reliably determine embryo ploidy has, therefore, increasingly been questioned in parallel. Methods We for that reason here established 2 mathematical models to assess probabilities of false-negative and false-positive results of an on average 6-cell biopsy from an approximately 300-cell TE. This study was a collaborative effort between investigators at The Center for Human Reproduction in New York City and the Center for Studies in Physics and Biology and the Brivanlou Laboratory of Stem Cell Biology and Molecular Embryology, the latter two both at Rockefeller University in New York City. Results Both models revealed that even under best case scenario, assuming even distribution of mosaicism in TE (since mosaicism is usually clonal, a highly unlikely scenario), a biopsy of at least 27 TE cells would be required to reach minimal diagnostic predictability from a single TEB. Conclusions As currently performed, a single TEB is, therefore, mathematically incapable of reliably determining whether an embryo can be transferred or should be discarded. Since a single TEB, as currently performed, apparently is not representative of the complete TE, this study, thus, raises additional concern about the clinical utilization of PGS

Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

<div><p>Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel <i>HTT</i> mRNA splice isoforms that are expressed in normal and <i>HTT</i>-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.</p></div

Human ES-derived motor neurons show increasing morphological complexity as they mature <i>in vitro</i>.

<p>(A) Top, schematic of ES cell directed differentiation to motor neurons shows timing of addition of the inductive cues retinoic acid (RA), and sonic hedgehog (SHH). Bottom, timing of morphometric and electrophysiological analyses. (B) Representative image of day 31+5 hESMN showing mature neuronal morphology and co-expression of GFP with motor neuron marker HB9. GFP intensity distinguished hESMN cell bodies (arrow, ∼65,000 gray levels (g.l.)), neurites (arrowhead, ∼18,000 g.l.), and cytoplasmic GFP background in non-MNs (star, ∼800 g.l.). Scale bar 50 µm. (C) Representative camera lucida (Metamorph) neurite traces from 5 randomly chosen (every 8^th) image fields at day 31+2, 31+5, and 31+9 show increasing neurite size and complexity. Scale bar 40 µm. (D-F) Soma area, branches, total neurite outgrowth and processes (not shown) were quantified (number of cells analyzed at each timepoint shown in brackets in D), median (grey line), mean (red line), 25–75 percentile (grey box), 10–90 percentiles (whisker bars), all outliers (+) are shown for each day from which measurements were made. The values for each morphometric parameter on each day were distributed non-normally (Shapiro-Wilk test, P<0.05) and Kruskal-Wallis One Way Analysis of Variance on Ranks showed significant changes in (D) cell soma area (H = 43.885, 2 d.f., P<0.001), (E) complexity or branches/cell H = 309.245, 2 d.f., P<0.001), (F) total neurite outgrowth (H = 161.287, 2 d.f., P<0.001), and (not shown) number of primary neurites (median, 25^th–75^th percentile: day 33: 3, 2–5; day 36: 6, 5–9; day 40: 8, 6–12, H = 442.555, 2 d.f., P<0.001). All significant <i>post hoc</i> pairwise comparisons, Dunn’s Method, are shown by black bars on graphs. and all pairwise comparisons for primary neurite number were significant, P<0.05.</p

Representative morphology and membrane potential responses to current step injection in hESMNs at 3 different times <i>in vitro</i>.

<p>Imaging of cells fixed after patch-clamp recordings indicate that recorded cells express the <i>Hb9</i>::GFP reporter transgene (A-C,E,G,and I). Voltage responses and imaging in the same rows are taken from same neurons. The neurons for A-C are same as that shown in F and G. D,F,H show examples of voltage responses to current steps recorded from 3 neurons current-clamped at −58 mV, −60 mV, and −55 mV, respectively. Bottom traces in D,F, and H show injected currents. Scale bars in images are 50 μm.</p

Spike frequency adaptation (SFA) and rebound action potentials (RAPs) in hESMNs.

<p>(A) An example of the change in instantaneous frequency during a train of action potentials evoked with positive current injection for 1 sec. Inset shows APs (upper) and injected currents (bottom). APs from which ‘a’ and ‘b’ ISI values were measure for SFA calculation are indicated. (B) SFA ratio, calculated as the maximum value of normalized ISIs after any amplitude of positive current injection, increased with DIV (n  = 8, R  = 0.73, P<0.05, Pearson’s linear regression). (C) RAPs were observed in a large subset of hESMNs. Upper trace shows voltage change after negative current injection. Bottom trace shows injected negative current steps. RAP follows the return of current to baseline after the hyperpolarizing step. (D) Incidence of RAPs in hESMNs at 4 different ages as indicated in Fig. 3 legend (n  = 29). Negative current steps with 5 pA increments (to at least 20 pA) were injected while checking for RAPs.</p

Developmental changes in intrinsic membrane properties of hESMNs.

<p>(A) Input resistance decreased over days <i>in vitro</i> (n  = 28, P<0.01, one-way ANOVA). ∗∗ P<0.01, Tukey’s <i>post hoc</i> test. (B) Resting membrane potential and (C) rheobase did not change (n  = 27 and 29, respectively). Positive current steps were injected in 5 pA increments to distinguish small differences in rheobase among individual neurons. (D) Half-width of action potentials (APs),, changed over time <i>in vitro</i> (n  = 26, P<0.001, one-way ANOVA). ∗∗ P<0.01, ∗∗∗ P<0.001, Tukey’s <i>post hoc</i> test. (E) Maximum frequency of APs after current injection increased over time <i>in vitro</i> (n  = 28, P<0.05, one-way ANOVA). ∗P<0.05, Tukey’s <i>post hoc</i> test. Dots shows frequency values for individual neurons. The numbers in parentheses indicate the number of neurons used for analysis taken from 22 dishes in total. In all panels, the first bar represents data from 31+2 DIV, 2^nd bar is 31+4 DIV, 3^rd bar is 31+8/31+9 DIV and 4^th bar is 31+12/31+13 DIV.</p

Time course of <i>HTT</i> isoform expression in hESCs differentiating to telencephalic neural fate.

<p>(A-D) RUES2 hESCs were differentiated to neural fate by blocking both branches of TGFβ signaling (default mechanism) as described in Materials and Methods. Values are normalized by GAPDH and displayed as fold change to day 0 values. Only the HTT-Δ10 isoform consistently decreases as the cells differentiate, while all three other isoforms maintain their expression levels unchanged. Error bars represent the standard error of the mean of 3 to 6 independent replicates. * p<0.05 vs d0.</p