Paper discussion

1. Conversion of Mouse and Human
fibroblasts into Functional
Spinal Motor Neurons
Esther Y. Son, Justin K. Ichida, Brian J. Wainger,
Jeremy S. Toma, Victor F. Rafuse, Clifford J. Woolf
and Kevin Eggan
Cell Stem cell; September 2, 2011
Presented By,
Dhwani Jhala
Graduate student
School of Life Science
Central University of Gujarat 1

2. Introduction
Pluripotent Stem Cells
Fibroblasts Motor Neurons
Differentiation Differentiation
Dedifferentiation
Transdifferentiation
Why Motor Neurons ?
• Motor neurons control the contraction of muscle fibers prompting movement
• Damage to MN caused by injury or disease can result in paralysis or death
• Interest in understanding how motor neurons regenerate after nerve injury and why
they are selective targets of degeneration in diseases such as spinal muscular
atrophy(SMA) and amylotrophic lateral sclerosis(ALS)
2

3. Why through transdifferentiation?
• Pluripotent stem cells may provide an inexhaustible reservoir but till date only
handful of neural subtypes have been produced this way and in many cases, they
have not been shown to possess refined, subtype-specific properties of particular
neurons
• Production of iMNs from iPSCs is very lengthy process and may take a year while
iMNs produced by transdifferentiation only takes few weeks which allows to test the
effect of new therapeutic very rapidly
• Direct reprogramming doesn’t involve use of any factors that are known to cause
cancer or any other diseased states.
• The most important thing is that iMNs function as normally as normal motor
neurons in in vitro and in vivo conditions
3

4. Objectives
1. Conversion of MEFs into MNs by providing specific transcription factors
2. Efficient production of iMNs by optimizing usage of transcription factors
3. iMNs possess a motor neuron gene expression signature
4. iMNs possess the electrophysiological characteristics of motor neurons
5. iMNs form functional synapses with muscle
6. iMNs integrate into the developing chick spinal cord
7. iMNs are sensitive to disease stimuli
8. Fibroblasts do not transit through a neural progenitor state before becoming iMNs
9. Human iMNs can be generated by eight transcription factors
4

5. Methods
• Isolation of embryonic and adult fibroblasts from mouse and humans
• Retroviral based transduction
• Obtaining ESC-derived and embryonic motor neurons
• FACS, Microarray Analysis, and qPCR
• Immunocytochemistry
• Electrophysiology : Whole-cell voltage-clamp and current-clamp techniques
• C2C12 Muscle Coculture
• In Ovo Transplantation of ESC-Derived Motor Neurons and iMNs
• Glia-Neuron Coculture for Disease Modeling
• Nestin::CreER Lineage Tracing
5

6. Conversion of MEFs into MNs by providing
specific transcription factors
Experimental outline: Eleven candidate transcription factors include eight
developmental genes in addition to the three iN factors
MEFs MN
6

7. (A) Hb9::GFP+ cells are generated from MEFs by transduction with 8 or 11 factors by day 35
post-transduction, but more efficiently by 11 factors. Scale bars represent 50 µm. (B) Hb9::GFP+
neurons express Tuj1 (purple) 7

8. Efficient production of iMNs by optimizing usage of transcription factors
(B) Efficiency of reprogramming when each factor is omitted from the 11-factor pool individually (C),
(D) iMNs generated with 10 factors (without Isl1) express endogenous Islet (red) Scale bar 40 µm in C
& 200 µm in D. 8

9. (E) Reprogramming efficiency is greater with Hb9 or Isl1 on top of four factors (Lhx3, Ascl1, Brn2, and
Myt1l) (F) Addition of Ngn2 to the six-factor pool (Hb9, Isl1, Lhx3, Ascl1, Brn2, and Myt1l) greatly
enhances reprogramming efficiency. (G) The seven iMN factors convert adult tail tip fibroblasts into
motor neurons. Scale bar represents 100 µm. 9

10. iMNs possess a motor neuron gene expression signature
(A) Global transcriptional analysis of FACS-purified Hb9::GFP+ motor neurons. (B–D) Pairwise gene
expression comparisons show that iMNs are highly similar to embryo-derived motor neurons and
dissimilar from the starting MEFs. 10

11. (A) iMNs express the panneuronal marker Map2 (red). Scale bars represent 100 µm. (B) iMNs express
synapsin (red). Scale bars represent 20 µm. (C) iMNs express vesicular choline acetyltransferase (vChAT,
red). Scale bars represent 40 µm. (D) iMNs express the motor neuron-selective transcription factor Hb9
(red). Scale bars represent 80 µm. 11

12. (E) qRT-PCR data showing expression of endogenous transcripts of the seven iMN
factors in iMNs and in ESC-derived motor neurons, relative to their levels in MEFs.
12

13. iMNs possess the electrophysiological
characteristics of motor neurons
Mechanism of action potential
Whole cell patch clamp technique
13

14. (A) iMNs express functional sodium channels. (B) iMNs express functional sodium and potassium
channels. (C) iMN sodium channel activity is appropriately blocked by tetrodotoxin (TTX). (D) iMNs
fire a single action potential upon depolarization. (E) iMNs fire multiple action potentials upon
depolarization. (F) 100 µM GABA induces inward currents in iMNs. (G) 100 µM glycine induces inward
currents in iMNs. (H) 100 µM kainate induces inward currents in iMNs. 14
Inward
currents
Outward
currents

15. iMNs form functional synapses with muscle
(Neuromuscular junctions- NMJs)
(I) iMN-induced contractions of C2C12 myotubes are blocked by 50 µM curare. The arrow indicates the
timing of curare addition. (J) iMNs cultured with chick myotubes form NMJs with characteristic a-
bungarotoxin (a-BTX, red) staining. The dotted line outlines the boundaries of a myotube. Scale bar
represents 5 µm. 15

16. iMNs integrate into the developing chick spinal cord
(A) Diagram showing the injection of iMNs into the neural tube of the stage 17 chick embryo.
(B) Transverse sections of iMN-injected chick neural tube 5 days after transplantation. Arrows in
both panels indicate the same axon of an iMN exiting the spinal cord through the ventral root. D:
dorsal, V: ventral, VR: ventral root.
16

17. iMNs are sensitive to disease
(Amyotrophic lateral sclerosis – ALS) stimuli
(C) FACS-purified Hb9::GFP+ iMNs cocultured with wild-type or the mutant SOD1G93A-overexpressing
glia for 10 days. Scale bars represent 5 µm. (D) Quantification of (C). (E) SOD1G93A iMNs exhibit
reduced survival in culture with wild-type glia. (F) Changes in iMN number after 9 days of culture in the
presence or absence of neurotrophic factors (GDNF, BDNF, and CNTF). 17

18. Fibroblasts do not transit through a neural progenitor
state before becoming iMNs
(A) Percentage of iMNs that have incorporated BrdU. (B) Outline of the lineage tracing experiment
using Nestin::CreER; LOX-STOP-LOX-H2B-mCherry; Hb9::GFP iPSCs or MEFs. To detect Nestin+
intermediates, cultures were treated with 1–2 µM 4-OHT during directed differentiation of iPSCs
(positive control) or during transdifferentiation of fibroblasts by the seven factors.
18

19. (C) FACS-purified, mCherry+ Hb9::GFP+ motor neurons derived from the triple transgenic iPSCs in the
presence of 1 µM 4-OHT. Expression of mCherry was observed in 3% of Hb9::GFP+ cells (n > 2,000) and
indicates the activation of Nestin::CreER during directed differentiation. Scale bars represent 40 µm.
(D) mCherry Hb9::GFP+ iMNs generated from the triple transgenic MEFs by transdifferentiation in the
presence of 2 µM 4-OHT. mCherry+ iMNs were never observed (n > 5,000), suggesting that a Nestin+
state is not accessed during reprogramming. 19

20. Human iMNs can be generated by eight transcription factors
(A) An Hb9::GFP+ neuron generated from a HEF culture by eight transcription factors(7+NEUROD1).
Scale bars represent 80 µm. (B) Quantification of human iMN reprogramming efficiency at day 30
posttransduction. (C) Human iMNs express vChAT (red). Scale bars represent 80 µm. 20

21. (D) Human iMNs express functional sodium and potassium channels. (E) Human iMNs fire action
potentials upon depolarization. (F) One hundred micromolars of kainate induces inward currents in
human iMNs. (G) One hundred micromolars GABA induces inward currents in human iMNs.
21

22. Conclusion
• The forced expression of selected transcription factors is sufficient to convert
mouse and human fibroblasts into induced motor neurons (iMNs).
• iMNs displayed a morphology, gene expression signature, electrophysiology,
synaptic functionality, in vivo engraftment capacity, and sensitivity to degenerative
stimuli similar to those of embryo-derived motor neurons.
• The converting fibroblasts do not transit through a proliferative neural progenitor
state before becoming motor neurons, indicating that they are formed in a manner
that is distinct from embryonic development.
• Thus, it can be concluded that iMNs are bona fide motor neurons.
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23. Critical Analysis
Strengths
• iMNs have been prooved as bonafide motor neurons by many ways.
•Production of iMNs is also prooved from human embryonic cells which gives stronger
impact to this experiment.
• The experiments are done in both in vitro and in vivo conditions indicating their
functional use in medical science.
Limitations
• There is possibility that other motor neuron-inducing factors have been overlooked
and varying them might enhance the frequency or accuracy of conversion.
• Conversion of human adult fibroblasts into motor neurons is not tested in this set of
experiments. If this is done in future then it would greatly facilitate the production of
patient-specific motor neurons for therapeutic uses in regenerative medicine and for
disease-related studies.
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