Theses Doctoral

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

Croft, Gist Fralley

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.



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More About This Work

Academic Units
Neurobiology and Behavior
Thesis Advisors
Henderson, Christopher
Ph.D., Columbia University
Published Here
January 27, 2012