Conversion of somatic cells into functional spinal motor neurons, and methods and uses thereof
The present invention provides methods of transdifferentiation of somatic cells, for example, directly converting a somatic cell of a first cell type, e.g., a fibroblast into a somatic cell of a second cell type, are described herein. In particular, the present invention generally relates to methods for converting a somatic cell, e.g., a fibroblast into a motor neuron, e.g., an induced motor neuron (iMN) with characteristics of a typical motor neuron. The present invention also relates to an isolated population comprising induced motor neurons (iMNs), compositions and their use in the treatment of motor neuron diseases such as ALS and SMA. In particular, the present invention relates to direct conversion of a somatic cell to an induced motor neuron (iMN) having motor neuron characteristics by increasing the protein expression of at least three motor-neuron inducing (MN-inducing) factors selected from Lhx3, Ascl1, Brn2, Myt1l, Isl1, Hb9, Ngn2 or NeuroD1 in a somatic cell, e.g., a fibroblast to convert the fibroblast to an induced motor neuron (iMN) which exhibits at least two characteristics of an endogenous motor neuron.
1. A method for producing a motor neuron from transdifferentiation of fibroblast, the method comprising increasing the protein expression of at least four motor-neuron inducing (MN-inducing) factors in the fibroblast, wherein the four motor-neuron inducing factors are Lhx3 and Ascl1 and two motor neuron inducing factors selected from the group consisting of: Brn2, Myt1l, Isl1, Hb9, Ngn2 or NeuroD, and wherein the motor neuron exhibits at least two characteristics selected from:
(i) the expression of at least two genes selected from the group consisting of: α2-tubulins, Map2, synapsins, synaptophysin, synaptotagmins, NeuroD, Isl1, cholineacetyltransferase (ChAT), or
(ii) functional characteristic of any of: ability to fire action potentials, produce an outward current in response to glycine, GABA or kainate, or produce an inward current in response to glutamate, and
wherein the fibroblast does not become a motor neuron progenitor prior to transdifferentiating into a motor neuron.
2. The method of claim 1 , wherein the four motor-neuron inducing factors do not include Myt1l or Brn2.
3. The method of claim 1 , wherein the four motor neuron inducing factors does not comprise miR-124.
4. The method of claim 1 , wherein the four motor neuron inducing factors does not comprise Isl1.
5. The method of claim 1 , wherein protein expression is increased by introducing into the fibroblast at least one nucleic acid encoding a MN-inducing factor.
6. The method of claim 1 , wherein the fibroblast is in vitro or ex vivo.
7. The method of claim 1 , wherein the fibroblast is obtained from a subject that has been diagnosed, or suspected as having a motor neuron disease or disorder, or has been identified to have a genetic predisposition linked with an increased risk of developing a motor neuron disease or disorder.
8. The method of claim 7 , wherein the motor neuron disease or disorder is amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA).