CREDIT SEMINARDirect conversion of

fibroblasts to functional neurons by

defined factors

CREDIT SEMINARDirect conversion of

fibroblasts to functional neurons by

defined factors

Major AdvisorDr. Gaya Prasad

By Syed Shafiq

Date: May 17, 2010. 2009V166M

Dept. of ABT

Major AdvisorDr. Gaya Prasad

By Syed Shafiq

Date: May 17, 2010. 2009V166M

Dept. of ABT

The research article has been The research article has been published in NATURE journal published in NATURE journal

dated feb.25, 2010 (vol 463) and is dated feb.25, 2010 (vol 463) and is authored by:authored by:

Thomas Vierbuchen, Austin Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Ostermeier, Zhiping P. Pang, Yuko

Kokubu1, Thomas C. Sudhof & Kokubu1, Thomas C. Sudhof & Marius Wernig Marius Wernig

IntroductionIntroduction

n The diverse cell types present in the adult organism are produced during development by lineage-specific transcription factors that define and reinforce cell-type-specific gene expression patterns.

n Cellular phenotypes are further stabilized by epigenetic modifications.

n Forced expression of lineage-specific genes in somatic cells can induce traits of other cell types.

n For example, the basic helix–loop–helix (bHLH) transcription factor MyoD (also called Myod1) can induce muscle-specific properties in fibroblasts but not hepatocytes.

A screen for neuronal-fate-

inducing factors

A screen for neuronal-fate-

inducing factors

Text

Transcription factors screened for neuron-inducing activity in MEFs

A pool of lentiviruses containing all 19 genes (19F pool) was prepared to infect mouse embryonic fibroblasts (MEFs) from TauEGFP knock-in mice, which express EGFP specifically in

neurons

Care was taken to exclude neural tissue from the MEF preparation, and the authors were unable to detect

evidence for the presence of neurons or neural progenitor cells in these cultures using

immunofluorescence, fluorescence activated cell sorting (FACS) and polymerase chain reaction with

reverse transcription (RT-PCR) analyses.

Representative Tuj1-positive cells 13 days after infection with Ascl1 alone or in combination with the indicated genes. Note the increased complexity of the neurites in the Ascl1+Myt1l

condition.

Uninfected p3 TauEGFP MEFs

19F pool, post 32 days infection

The effect of 18 transcription factors in combination with Ascl1 on neuronal

induction 13 days post infection

5F pool, 13 days post infection

5F pool, 22 days post infection

iNs derived from Balb/c MEFs

Characterization of 5-factor

iN cells

Characterization of 5-factor

iN cells

n Patch-clamp recordings of TauEGFP-positive cells on days 8, 12 and 20 after infection, were performed.

n For the majority of the iN cells analysed (85.1%), action potentials could be elicited by depolarizing the membrane in current-clamp mode.

n Six cells (14.2%) showed spontaneous action potentials, some as early as 8 days after transduction.

n The resting membrane potentials ranged between -30 and -69 mV with an average of , -55 mV on day 20.

n Induced neuronal cells responded to exogenous application of GABA, and this response could be blocked by the GABA receptor antagonist picrotoxin.

n Thus, MEF-derived iN cells seem to exhibit the functional membrane properties of neurons and possess ligand-gated GABA receptors.

K-CURRENT CLAMP MODE; L-VOLTAGE CLAMP; M-SPONTANEOUS AP

5F pool, 22 days post infection

Characterization of neurotransmitter phenotype of iNs

Some iN cells (9 out of ,500) contained the Ca2+binding protein calretinin, a marker for cortical interneurons and other neuronal subtypes.

No expression of tyrosine hydroxylase, choline acetyltransferase or serotonin was detected

The vast majority of iN cells were negative for peripherin, an intermediate filament characteristic of peripheral neurons

Functional neurons from tail fibroblasts

Functional neurons from tail fibroblasts

n Tail-tip fibroblasts (TTFs) from 3-day-old TauEGFP and Rosa26-rtTA mice were isolated.

n Pre-existing neurons, glia, or neural progenitor cells could not be detected in the cultures.

n Electrophysiological recordings 12 days after infection demonstrated an average resting membrane potential of ,-57 mV (range: -35 to -70 mV), firing of action potentials (81.8%) and expression of functional voltage-gated membrane channel proteins.

n Tyrosine hydroxylase, choline acetyltransferase, or serotonin expression was not detected.

5F pool, post 13 days post infection

5F pool, 21 days post infection

Nine of eleven cells recorded exhibited action

potentials

Neuronal induction is

fast and efficient

Neuronal induction is

fast and efficient

n The authors assessed the kinetics and efficiency of 5F iN conversion.

n Tuj1-positive cells with immature neuron-like morphology were found as early as 3 days after infection.

n After 5 days, neuronal cells with long, branching processes were readily detected, and over time increasingly complex morphologies were evident.

n TauEGFP expression was detected as early as day five.

n Electrophysiological parameters also showed signs of maturation over time.

n To estimate the conversion efficiency, they first determined how many of the MEF-derived iN cells divided after induction of the viral transgenes by treating the cells with 5-bromodeoxyuridine (BrdU) throughout the duration of the culture period and beginning 1 day after gene induction.

n Vast majority of iN cells became post mitotic by 24 h after transgene activation.

n The efficiency ranged from 1.8% to 7.7% in MEF and TTF iN cells presumably due to slight variations in titres of the viruses.

Cells were maintained at a potential of approximately -65 to -70 mV. Step current injection

protocols were used from -50 to +70 pA

BrdU-positive iN cells after BrdU treatment from day 0–13 or day 1–13

after transgene induction.

5F pool, 5 days post infection 13 days post infection

iN cells form functional synapses

iN cells form functional synapses

n Two independent methods were used.

n First, they determined whether iN cells were capable of synaptically integrating into pre-existing neural networks.

n TauEGFP-positive iN cells were FACS purified 7–8 days after infection of MEFs and plated on cortical neuronal cultures (7 days in vitro).

n One week after re-plating, patch-clamp recordings from TauEGFP-positive iN cells were performed.

n Spontaneous and rhythmic network activity typical of cortical neurons in culture, was observed.

n Both EPSCs and IPSCs could be evoked after electrical stimulation delivered from a concentric electrode placed 100–150 mm away from the patched iN cell.

n In the presence of the a-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid (AMPA) and NMDA (N-methyl-D-aspartate) receptor channel blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonovaleric acid (D-AP5), spontaneous IPSCs were reliably detected.

n Moreover, synaptic responses recorded from iN cells showed signs of short-term synaptic plasticity, such as depression of IPSCs and facilitation of EPSCs during a high-frequency stimulus train.

n The presence of synaptic contacts between iN cells and cortical neurons was independently corroborated by the immunocytochemical detection of synapsin-positive puncta surrounding MAP2-positive dendrites originating from EGFP-positive cells.

n Similar experiments performed with iN cells derived from TTFs also resulted in synaptic responses.

n Thus iN cells can form functional postsynaptic compartments and receive synaptic inputs from cortical neurons.

iNs from TTF

n Next they asked whether iN cells were capable of forming synapses with each other.

n They plated FACS-sorted TauEGFP-positive, MEF-derived 5F iN cells 8 days after infection onto a monolayer culture of primary astrocytes.

n These cultures were confirmed to be free of pre-existing Tuj1- or MAP2-positive neurons.

n Patch-clamp recordings at 12–17 days after sorting indicated the presence of spontaneous postsynaptic currents in 5 out of 11 cells.

n Upon extracellular stimulation, evoked EPSCs could be elicited in a majority of the cells (9 out of 11 cells).

n Both NMDA-receptor-mediated (9 out of 11 cells) and AMPA-receptor-mediated EPSCs (8 out of 11 cells) were recorded.

n They were unable to detect obvious IPSCs in a total of 15 recorded 5F iN cells.

NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl- benzo[f]quinoxaline-2,3-dione) is an AMPA-receptor antagonist

These data indicate that iN cells are capable of forming functional synapses with each other, and that

the majority of iN cells exhibit an excitatory phenotype.

MEF-derived 5F-iN cells on glia express markers of glutamatergic neurons

Genes sufficient for

neuronal conversion

Genes sufficient for

neuronal conversion

n The authors next attempted to determine the relative contribution of each of the five genes by removing each gene from the pool and assessing the efficiency of iN cell generation.

n Only the omission of Ascl1 had a marked effect on induction efficiency.

n Thus, they tested the effects of removing two genes at a time.

n Either Ascl1 or both Brn2 and Myt1l must be present to generate iN cells.

n Most efficient conversions were achieved when Ascl1 and Brn2 were combined with either Myt1l (BAM pool) or Zic1 (BAZ pool).

n MEF-derived BAM iN cells expressed the pan-neuronal markers MAP2 and synapsin.

n The BAM pool was capable of efficiently generating iN cells from perinatal TTFs.

n When co-cultured with astrocytes, both MEF and perinatal TTF-derived BAM iN cells were capable of forming functional synapses as determined by the presence of both NMDA- and AMPA-receptor mediated EPSCs.

n Majority of BAM iN cells were found to be excitatory.

n 53% (111 out of 211 cells) of MEF BAM iN cells expressed Tbr1, a marker of excitatory cortical neurons, whereas less than 1% (3 out of 500 cells) were GAD-positive.

n These results left open the possibility that one or two factors might be able to induce functional neuronal properties in MEFs.

13 days post infection

12 days post infection

12 days post infection

BAM pool, 22 days post infection

BAM pool, perinatal TTF derived iNs, 12d post

infection

BAM pool, 21d post infection

BAM pool, 22d post infection

BAM iN cells derived from TTF isolated from a six-week-old TauEGFP mouse

Patch-clamp recordings for

BAM iNs showing synaptic

formation

53% cells were Tbr1 +ve

only 1% are GAD +ve

immature responses from Ascl alone

Thus, it seems likely that Ascl1 alone is sufficient to induce some neuronal traits, such as expression of

functional voltage-dependent channel proteins that are necessary for the generation of action potentials;

however, co-infection of additional factors is necessary to facilitate neuronal conversion and maturation.

Discussion

Discussion

n Expression of three transcription factors can rapidly and efficiently convert mouse fibroblasts into functional neurons (iN cells).

n Although the single factor Ascl1 was sufficient to induce immature neuronal features, the additional expression of Brn2 and Myt1l generated mature iN cells with efficiencies of up to 19.5%.

n Three-factor iN cells displayed functional neuronal properties.

n The highly efficient nature of this process effectively rules out the possibility that directed differentiation of rare stem or precursor cells with neurogenic potential can explain our observations.

n Future studies need to be performed to demonstrate that terminally differentiated cells can be directly converted into neurons and to decipher the molecular mechanism of this fibroblast to neuron conversion.

n It is possible that certain subpopulations of cells are ‘primed’ to respond to these factors, depending on their pre-existing transcriptional or epigenetic states.

n The majority of iN cells described in this report are excitatory and express markers of cortical identity.

n A low proportion of iN cells expressed markers of GABAergic neurons, but no other neurotransmitter phenotypes were detected.

n Additional combinations of neural transcription factors might also be able to generate functional neurons whose phenotypes remain to be explored.

n One of the next important steps will be to generate iN cells of other specific neuronal subtypes and from human cells.

n Future studies will be necessary to determine whether iN cells could represent an alternative method to generate patient-specific neurons.

n The generation of iN cells is fast, efficient and devoid of tumorigenic pluripotent stem cells, a key complication of induced pluripotent stem cell approaches in regenerative medicine.

n Therefore, iN cells could provide a novel and powerful system for studying cellular identity and plasticity, neurological disease modelling, drug discovery and regenerative medicine.

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