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March 24 2015 Final Report

Investigating cellular reprogramming as a tool for identifying

neural based assays for post-traumatic stress disorder (PTSD)

Prepared By: James Ellis, PhD – Hospital for Sick Children

Contractor: James Ellis, Hospital for Sick Children Peter Gilgan Centre for Research and Learning 686 Bay Street, 16th Floor - Room 9715, Toronto, Ontario, M5G 0A4 Tel: 416-813-7295 Task #27 under contract W7714-125624/001/SV

Contract Scientific Authority: Henry Peng, Defence Research and Development Canada Defence Scientist/Individual Behaviour and Performance

The scientific or technical validity of this Contract Report is entirely the responsibility of the Contractor and the contents do not necessarily have the approval or endorsement of the Department of National Defence of Canada. Contract Report DRDC-RDDC-2015-C095 March 2015

© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2015

© Sa Majesté la Reine (en droit du Canada) telle que représentée par le ministère de la Défense nationale, 2015

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TABLE OF CONTENTS

Executive Summary…………………... 3

Introduction…………………………… 4

Tasks Completed……………………… 5

Conclusion…………………………….11

Proposal for Future Work……………..11

References…………………………….13

LIST OF FIGURES AND TABLES

Table 1. Summary of current iNPC lines and their characterization….10

Figure 1. Direct conversion of NPCs from human fibroblasts….15

Figure 2. Time-course expression of pluripotent genes during direct conversion from human

fibroblasts indicates that iNPCs may not pass through an iPSC intermediate stage…16

Figure 3. Immunocytochemical analysis with multiple markers demonstrate highly pure

preparations of iNPCs….16

Figure 4. Examination of regional identity of iNPCs using quantitative gene expression analysis

shows dorsal-hindbrain identity….17

Figure 5. Single-step direct conversion of mature neurons from hPSCs….17

Figure 6. Quantitative analysis of iNPC-derived neurons differentiated by a single-step protocol

demonstrates highly pure neuron production….18

Figure 7. Immunocytochemical analysis with multiple markers demonstrates tripotency of

iNPCs (the ability to generate all three neural lineage cells)….18

Figure 8. Generation and characterization of iNPCs from healthy adult fibroblasts….19

Figure 9. Generation and characterization of iNPCs from RTT patient derived fibroblasts….20

Figure 10. Quantitative gene expression analysis showing the RTT-iNPC lines do not express

WT MECP2 transcript….20

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EXECUTIVE SUMMARY

Our goal is to model neuropsychiatric disorders including PTSD by developing new

methods to generate patient neurons to study in vitro. Many human diseases have now been

modeled in vitro by reprogramming somatic cells (usually skin fibroblasts) into stem cells that

can then be used to generate the affected cell type. This is usually accomplished by

reprogramming somatic cells into induced Pluripotent Stem cells (iPSC). However, iPS cells

erase epigenetic memory and therefore may not be suitable for modeling stress-induced disorders

such as PTSD. In contrast, the ability to reprogram skin cells directly into Neural Progenitor

Cells (NPC) is predicted to maintain the epigenetic modifications induced by stress. This

technology should also permit more rapid generation of neurons from the patient sample than

using iPSC. To this end, we evaluated and confirmed that gene transfer of 4 reprogramming

factors using retrovirus vectors into a human foreskin cell line (BJ), in combination with several

small molecules, does in fact lead to the formation of induced NPC colonies (iNPC). We isolated

and expanded these colonies into iNPC lines and demonstrated they gradually mature over 20

passages and expressed markers of dorsal hindbrain. The iNPCs were tripotent and generated

neurons, astrocytes and oligondendrocytes as expected over 11 weeks. We also employed a

single step conversion protocol to more rapidly direct the differentiation of iNPCs into cortical

neurons over 3 weeks by transient expression of NGN2 in a lentivirus vector. Additional

experiments showed that the induction of iNPCs was reproducible on BJ cells, as well as healthy

adult human fibroblasts and fibroblasts derived from an individual with Rett syndrome. We are

currently expanding these iNPC lines to attempt to model Rett syndrome phenotypes in

hindbrain neurons, which we have previously done using patient iPSC derived cortical neurons.

Overall, we accomplished our goal to generate iNPC from human fibroblasts and differentiate

them into neurons. We propose that short-term future work should focus on completing the

phenotyping of the Rett syndrome neurons. The long-term future goal is to modify iNPC

generation by employing Sendai virus vectors to reprogram patient peripheral blood, which is

more easily acquired than skin biopsies. The ability to rapidly generate PTSD patient neurons in

vitro will provide an important new system to evaluate the disorder.

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INTRODUCTION

Post-traumatic stress disorder (PTSD) is a stress-related disorder that develops after

exposure to intense trauma. Over half of the population will be exposed to a psychologically

traumatic event at some point in their life, with approximately 10-20% moving on to develop

PTSD1. Accumulating evidence indicates that the alteration of proper neuronal structure and

function in the cortex via altered gene expression underlies the pathophysiology of PTSD2-5.

However, the lack of appropriate animal model systems and/or the difficulty of obtaining human

brain tissue have been a major hurdle to evaluating and interpreting these findings. The advent of

induced pluripotent stem cell (iPSC) technology and advanced neural differentiation techniques

has allowed for the detailed functional analyses of personalized neurons6. Generation of neurons

from patients with neurological disorders provides a great opportunity for studies of neuronal

populations that may be impaired in the disease. Moreover, recently developed technologies for

direct conversion of neurons or neural progenitor cells (NPCs) from patient’s somatic cells,

allows the generation of personalized neurons in an unprecedented fast and efficient manner.

These approaches may pave a way for identification of predisposing genetic factors, discovery of

relevant biomarkers for susceptibility and ultimately the development of preventative and

therapeutic interventions. In this study, we seek to develop methods to efficiently generate

neuronal cells from individuals with PTSD using cell reprogramming technology. In particular,

we will focus on several scalable approaches, including a method for direct conversion of human

cells into NPCs, and a single step protocol to convert human NPCs into neurons. The current

state of reprogramming methods is summarized in detail in the literature review provided to

DRDC in 2014.

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TASKS COMPLETED

5.1 Perform a literature review of neural differentiation from stem cells for

neuropsychiatric research. Complete.

The literature review written by D.S. Kim and J. Ellis was submitted to DRDC, and a

modified version was published in Frontiers in Cellular Neuroscience 8:109 2014.

5.2 Develop methods for the direct conversion of specific human cells into Neural

Progenitor Cells (NPCs). Complete.

In the quarterly progress reports submitted date, we described an optimal procedure for

reprogramming NPCs from human fibroblasts (Figure 1). Our results showed that addition of

signaling molecules (human leukemia inhibitory factor and basic fibroblast growth factor) and a

cocktail of small molecules (a WNT signal activator (CHIR99021, CHIR), an Activin/Nodal

signal inhibitor (SB431542), a Rho-kinase/ROCK inhibitor (Thiazovivin) and a BMP signal

inhibitor (LDN193189)) facilitated the formation of putative NPC colonies from human neonatal

fibroblasts (BJ fibroblasts) reprogrammed by retroviral delivery of the OSKM transcription

factors (OCT4, SOX2, KLF4, and c-MYC). Out of 17 NPC like-colonies that were isolated

mechanically, six colonies were successfully expanded in the same culture condition, and two of

them (referred to as iNPC-B2 and B6) were subjected to detailed characterization. The cells

showed marker expression of NPCs, and maintain self-renewal without losing the feature of

NPCs over multiple passages. Moreover, the iNPCs showed normal karyotypes up to passage 25,

although Southern blot analysis revealed they shared the same retrovirus integration sites

demonstrating that they were subclones of each other.

To ask whether the iNPCs were the result of direct conversion of fibroblasts or

spontaneously differentiated progeny of iPSCs that arise by chance, we examined time-course

expression of pluripotent markers such as NANOG and REX1 during conversion after viral

infection of the 4-factors. Although there was a slight increase of NANOG expression at 4 weeks

after infection, REX1 expression was not detected at all (Figure 2). This result supports the

likelihood that iNPCs were derived by lineage conversion rather than spontaneous differentiation

from pluripotent cells.

Previous studies have claimed that lineage conversion is a progressive process7. This

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notion prompted us to examine whether the molecular signature of iNPCs could change and

become closer to that of authentic NPCs during ongoing passage. Our result supports the idea

that lineage conversion occurs progressively and that the iNPCs need to be passaged roughly 20

times. To examine the purity of the cultures, we performed immunostaining with NPC markers

and quantified the number of positive cells. At passage 20, the majority of cells in the culture

were highly positive for all NPC markers tested: Almost 100% cells were NESTIN-positive, and

over 80% of cells were positive for SOX1, PAX6, and PLZF, which are definitive neural

markers (Figure 3). Given the fact that the most efficient protocol for neural differentiation of

hESCs so far gives rise to around 80% of SOX1 positive cells, this result impressively shows

that this culture condition generates a highly pure population of NPCs.

It is known that NPCs retain a particular regional identity within the entire central

nervous system (CNS), which means that NPCs exhibit the specific molecular profile depending

on which region of the CNS from which they originate8. The regions are designated by the

anatomical positions known as the anterior-posterior (A-P) axis and dorsal-ventral (D-V) axis. It

is also suggested that the regional identity of NPCs underlies the determination of cell fate

during development9. It is critical information to know the regional identity because it

determines the fate or subtype of the neurons that can be produced.

In previous reports, we examined four markers for anterior-posterior identity, but we

added two more markers (PAX2 for midbrain, GBX2 for hindbrain) to finely determine their

regional identity at A-P axis. Consistent with our previous result, quantitative gene expression

revealed that iNPCs predominantly showed posterior fate, in particular of hindbrain (GBX2 and

HOXB4), at the A-P axis (Figure 4 left). All iNPC lines showed higher expression of hindbrain

markers than those of forebrain and midbrain. Since the result indicated that iNPCs are from the

hindbrain, we tested the expression of D-V markers that are known to be specific for the

hindbrain domain (IRX3 and NKX6.1 for dorsal hindbrain, NKX2.2 and OLIG2 for ventral

hindbrain). All three different cell lines tested exclusively showed expression of dorsal markers

(Figure 4 Right). Therefore, our result suggested that iNPCs generated by this method are of

dorsal hindbrain identity. These results are expected because the culture condition contains

GSK3b inhibitor, which is often used as an activator of canonical wnt pathway, and activation of

WNT signal has been known to be implicated in posteriorization and dorsalization in developing

brain10. We therefore predict that neurons generated from the NPCs should be of hindbrain origin,

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and these could be useful for studying certain disorders such as Rett syndrome which has

hindbrain phenotypes related to breathing.

5.3 Develop a single step protocol to convert human NPCs into neurons. Complete.

We have explored the possibility of utilizing the single step conversion protocol for in

vitro disease modeling11 (Figure 5). Since we found that differentiation of iNPCs to functional

and mature neurons takes a relatively long period of time (more than 8 weeks), the single step

conversion protocol by forced expression of NGN2 may be a good alternative for obtaining

functional neurons in a shorter period of time and with better efficiency. Most importantly,

NGN2 overexpression forces iPS cells to differentiate into well defined and functional cortical

neurons. We have continued to optimize the single step conversion protocol by forced expression

of NGN2 in iNPCs. In previous reports, we have shown that Dox-induced overexpression of

NGN2 for a week forces iNPCs to differentiate into MAP2-positive cells with mature neuronal

morphology in just three weeks. In an effort to determine the neuronal differentiation efficiency

of this approach, we quantified the numbers of MAP2 and NeuN-positive cells among hNA-

positive human cells within the culture. As shown in Figure 6, the majority of human cells were

positive for MAP2 (83% among total cells) and NeuN (78% among MAP2-positive cells)

indicating that the single-step conversion method by NGN2-overexpression enables iNPCs to

differentiate rapidly into mature neurons with high efficiency.

5.4 Prepare a progress report on tasks 5.1-5.3. Completed for all previous quarters.

5.5 DRDC, Toronto Research Centre will prepare a study protocol for collection and

analysis of tissue samples from PTSD patients and healthy controls. This task was revised by

DRDC and healthy and Rett syndrome fibroblast samples were used from SickKids instead for

Task 5.8.

5.6 Develop in vitro assays to assess neural functions. Using the standard differentiation

method described in Task 5.7 below, we previously tested whether iNPC-derived neurons exhibit

functional membrane properties. Whole-cell patch-clamp recordings were performed through a

colaboration with Dr. Michael Salter’s group at Sickkids Hospital. Current clamp recordings

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show that the neurons generated repetitive trains of action potentials. The voltage clamp mode

recordings show rapidly inactivating inward sodium current and persistent outward potassium

currents in response to depolarizing voltage steps. However, we could not detect the neurons

exhibiting spontaneous excitatory postsynaptic currents (EPSCs), implying that the probability of

functional synaptic connections may be quite low. This connectivity may be improved if the

neurons are cultured on astrocytes in future experiments.

To further examine whether there were functional synapse formations among

differentiated neurons, we stained the neurons with synaptic marker proteins such as synapsin-1

(a pre-synaptic protein), PSD-95 and SHANK2 (post-synaptic proteins) along with MAP2.

Interestingly, we were able to detect strong co-localization among pre- and post-synaptic proteins

along the MAP2-positive dendrites, but these were rare. These results suggest that even though

the neurons could form functional synaptic connections with each other, they may still be

immature for intensive synapse formation. Collectively, our data demonstrate that iNPCs could

give rise to neurons displaying functional membrane potentials and synaptic connectivity. Given

the fact that neurons of many neuropsychiatric disorders show a defect in synaptic connectivity,

neurons that are fully differentiated will be required to verify whether iNPC-derived neurons

form functional synapses.

5.7 Characterize the neuronal types generated above. To investigate the differentiation

potential of the iNPCs we determined whether they can give rise to neurons, astrocytes, and

oligodendrocytes under appropriate conditions. To induce spontaneous differentiation of iNPCs

toward the three neural lineages, we cultured them (iNPCs-B2, at passage 10) in the presence of

0.5% fetal bovine serum12. After two weeks of differentiation, the cells began to exhibit an

immature neuronal morphology with two or three long processes. Immunostaining showed that

many of them were indeed immature neurons positive for double-cortin 2 (DCX2) and/or βIII-

tubulin. After 8 weeks of differentiation, we were able to observe VGluT1-positive (excitatory),

GABA-positive (inhibitory), and TH-positive (catecholaminergic) neurons as well as GFAP-

positive astrocytes. Inspired by the fact that oligodendrocyte precursors appear at later stages of

mammalian neural development than neurons or astrocytes13, we have further differentiated

iNPCs for a longer time than it requires for differentiating neurons and astrocytes, and found that

O4-positive oligodendrocyte precursors were detected at 11 weeks after differentiation (Figure 7).

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This result clearly shows that the iNPCs are tripotent neural stem cells that can generate all three

neural lineage cells (neurons, astrocytes and oligodendrocytes).

5.8 Convert healthy and patient tissue samples to NPC.

Finally, as a proof-of-concept for disease modeling, and to validate the robustness of our

lineage conversion method, we attempted to generate iNPCs from fibroblasts obtained from a

Rett syndrome (RTT) patient as well as from a healthy adult donor. Retroviral infection of

OSKM transcription factors and exposure to cocktails of signaling molecules and small

molecules led the fibroblasts to undergo drastic morphological changes as described before.

Because the BJ fibroblasts that we initially used to establish our method were derived from

newborn foreskin, we first tested whether our method can reprogram the adult healthy male

(SK0019-002) cells. Reassuringly, our method successfully derived two stable iNPC lines and

immunocytochemistry revealed that many cells were positive for multiple neural markers tested,

and there was no NANOG-positive cells detected (Figure 8). Therefore, the method is able to

generate iNPCs from human fibroblasts from both newborns and adults.

The ultimate goal of the project is to explore the possibility that direct conversion can

provide an in vitro modeling for neuropsychiatric disorders. With encouragement from the

DRDC we generated iNPCs from a patient with RTT, a severe neurodevelopmental disorder

caused by mutation in the methyl-CpG binding protein 2 (MECP2) gene. A successful round of

direct conversion was conducted on RTT-fibroblasts that have an MECP2 deletion from exon 3

to exon 414, and we established 2 iNPC lines. These RTT-iNPCs were highly positive for Nestin,

SOX1, SOX2, and PAX6 (Figure 9). They were also highly proliferative judging by the high

percentage of ki67-positive cells in the culture. To test whether the iNPCs derived from RTT-

fibroblasts are in fact mutant cells, we examined quantitative gene expression of MECP2. Indeed,

we found that RTT-iNPCs express extremely low levels of WT MECP2 transcript (Figure 10),

suggesting that they are most likely all mutant cells. We are currently differentiating them into

neurons to determine whether the hindbrain neurons exhibit disease-related phenotypes that are

displayed in cortical neurons derived from RTT-iPSC14,15. This experiment will give us an

insight into the possibility of using this technology as a disease model for neuropsychiatric

disease.

The iNPC lines generated to date and their current state of characterization are

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summarized in Table 1. Since the wild-type BJ iNPC lines in the first experiment were shown

to be identical subclones, we initiated the direct conversion process for a 2nd batch of BJ

fibroblasts. The 15 best colonies were picked and expanded to establish at least 1 additional

purified BJ clonal cell line. We will then have 2 clonal lines generated from each of the 3

individual fibroblast samples for disease modeling experiments. iNPCs are being purified and

expanded for two control samples (BJ and SK0019-002) and one mutant sample (RTTΔ3-4).

Characterization has been finished for the majority of the clonal lines to show that the iNPCs

express precursor markers (SOX1, SOX2, Nestin and PAX6) and are proliferative (Ki67).

Characterization will be completed for remaining lines and frozen vials of cells will be made to

ensure a stable supply of cells for further experiments. Different individuals are currently

working with the cells to demonstrate the protocol’s reproducibility and robustness between

multiple users.

Table 1 – Summary of current iNPC lines and their characterization

Genotype Cell Line & Clone # iNPC Characterization Frozen Vials

Wild-type

BJ #2 (1st batch) ✔ ✔ Note: #2 & #6

are the same

clone BJ #6 (1st batch) ✔ ✔

Wild-type SK0019-002 #5 ✔ Purifying

SK0019-002 #10 In Progress Purifying

MECP2-null RTTΔ3-4 #10 ✔ Purifying

RTTΔ3-4 #42 ✔ Purified, expanding

Wild-type BJ #1-15 (2nd batch) Picked 15 colonies;

establishing clonal lines -

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CONCLUSION

We accomplished our goal to generate iNPC from human fibroblasts and differentiate

them into neurons. We expect to have established 2 iNPC lines from each of 2 healthy

individuals and a Rett syndrome patient. These lines require 20 passages to mature into iNPCs

that have a dorsal hindbrain specificity. The iNPCs produce immature neurons by 8 weeks of

differentiation and may become more functional by coculture with astrocytes. Importantly, the

one step conversion protocol forces the iNPCs to differentiate into human neurons in just 3

weeks, and earlier reports indicate these should have a cortical identify and be more mature

because they are plated on astrocytes. We are currently expanding the iNPC lines in order to

determine the phenotype of Rett syndrome hindbrain neurons as a proof-of concept that the iNPC

reprogramming method can be used to model neuropsychiatric disorders such as PTSD.

PROPOSAL FOR FUTURE WORK

Short-Term Goal: iNPC disease modeling of Rett syndrome neuron phenotypes

The short-term goal of the project is to establish a proof-of-concept that the direct

conversion of iNPCs can be an effective method for neuropsychiatric disease modeling. In the

absence of PTSD samples, we are performing studies on Rett Syndrome iNPCs. Characterization

of the iNPCs indicates regional specificity to the dorsal hindbrain, likely giving rise to neurons of

the same fate. Rett Syndrome patients can experience autonomic abnormalities, including

disrupted respiratory and heart function. This is likely due to loss of MECP2 in the hindbrain, the

area of the brain important for vital autonomic processes. This was shown in an RTT mouse

model where Mecp2 is removed from the brainstem and spinal cord, resulting in poor respiratory

and heart function and decreased life span in the mice16. Thus we now aim to differentiate

passage 20 iNPCs into hindbrain neurons and study disease-related phenotypes such as smaller

soma/nuclear size, reduced dendritic complexity, and defects in electrophysiological properties

similar to iPSC-derived cortical neurons. These experiments will be the first to show that iNPCs

can be used to model human disease and will be the first exploration of RTT hindbrain neuron

phenotypes using reprogrammed cells. The outcomes of this study will provide us with important

insight into neurons obtained by direct lineage conversion and could serve as an alternative in

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vitro model system to investigate the mechanisms underlying onset and manifestation of the

various human neuropsychiatric diseases including PTSD.

Several issues need to be addressed to successfully model disease in vitro using the

iNPC protocol. These limitations include: 1) the hindbrain specificity of the iNPCs, 2) the

requirement for extensive passage for the acquisition of NPC properties, and 3) the lack of

evidence that the iNPCs are equivalent to authentic NPCs in vivo. We will address these concerns

in two ways. Our lab and other groups have previously characterized cortical excitatory neurons

from RTT iPSCs. With the healthy and RTT iNPC lines we have generated, we propose to first

differentiate them into cortical excitatory neurons for morphological phenotyping using the

single-step conversion method. This involves the forced expression of the neuronal lineage-

specific transcription factor NGN2 in the iNPCs for 1 week to generate highly pure populations

of cortical excitatory neurons. We will determine if the RTT neurons derived from NGN2

transduced iNPCs express cortical markers, have a smaller soma/nuclear size and

dendritic/electrophysiologic defects to recapitulate our findings when modeling with neurons

derived from iPSCs. This would demonstrate that cortical neurons can be derived more quickly

using the single step method on iNPCs, and that our method is valid for neuropsychiatric disease

modeling studies.

To convincingly demonstrate that the iNPC lines resemble their in vivo counterparts, we

are using other funding sources to determine whether they survive and differentiate in mouse

brains. In vivo transplantation experiments can provide us with profound information about the

functionality and differentiation potential of the iNPCs. Integration of engrafted cells into host

tissue reflects the functionality of the cells, and differentiation to three neural lineages after

transplantation shows its multipotency. We have transplanted iNPCs labeled with EGFP into

neonatal mouse brain, and will assess the engraftment and differentiation of transplanted cells in

host brain using immunocytochemistry for hNA and EGFP at 8-16 week post-transplantation.

Overall, these short term goals will demonstrate that the iNPC direct conversion method can be

used to model a neuropsychiatric disorder in hindbrain and cortical neurons.

Long-Term Goal: iNPC generation from blood to model cortical neuron phenotypes

The long-term goal of this project is to reprogram blood cells from PTSD patients in a

way that preserves the stress-induced alterations and allows rapid differentiation into cortical

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neurons to identify differences from unaffected controls. To this end, an REB to collect

peripheral blood for reprogramming has been submitted at SickKids. We know that Sendai virus

is very efficient at delivering the 4 reprogramming factors into blood cells17. We propose to

modify our iNPC direct conversion protocol for use on human blood. Blood samples will be

infected with the SeV reprogramming virus and treated with the chemical compounds to promote

iNPC colony formation. The colonies will be picked and established into iNPC lines. The

effectiveness of the single step NGN2 differentiation procedure will be determined on these new

iNPCs and its ability to generate functional cortical neurons tested. These experiments will

culminate in a pipeline capable of producing neurons from patients with neuropsychiatric disease.

At this point we would seek permission to reprogram PTSD or other patient blood into iNPCs for

disease modeling in cortical neurons and ultimately for drug testing.

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7. Han D. W. et al., Direct reprogramming of fibroblasts into neural stem cells by defined

factors. Cell Stem Cell. 10, 465-472. (2012).

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8. Guillemot F. Cellular and molecular control of neurogenesis in the mammalian telencephalon.

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Neurosci. 11, 77-86. (2010)

11. Zhang Y., et al. Rapid single-step induction of functional neurons from human pluripotent

stem cells. Neuron.78, 785-798. (2013).

12. Kim D. S. et al., Highly pure and expandable PSA-NCAM-positive neural precursors from

human ESC and iPSC-derived neural rosettes. PLoS One. 7, e39715. (2012).

13. Gallo V., and Deneen B. Glial development: the crossroads of regeneration and repair in the

CNS. Neuron. 83, 283-308. (2014).

14. Cheung A. Y., et al. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic

controls through X-chromosome inactivation. Hum Mol Genet. 20, 2103-2115. (2011).

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Figure 1. Direct conversion of NPCs from human fibroblasts. (A) Schematic of direct

conversion of NPCs from human fibroblasts. (B) 5 days after retroviral infection (OSKM), a few

fibroblasts showed morphological changes (more compact and oval shape, indicated by arrow

heads) distinct from surrounding fibroblasts, in the presence of two small molecules (2.5μM

SB431542 and 3μM CHIR99021) and LIF in CDM media. (C) Addition of 20ng/ml bFGF to

neural inducing media remarkably enhances the number of cells with morphological changes

described above (indicated by dotted lines). (D) After an additional 2 weeks, Nestin-positive

NPC-like cells appeared in the culture. (E) iPSC-like colonies were frequently observed after one

week of the direct conversion procedure (indicated by a yellow arrow).

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Figure 2. Time-course expression of pluripotent genes during direct conversion from

human fibroblasts indicates that iNPCs may not pass through an iPSC intermediate stage.

NANOG and REX1 are only expressed in pluripotent stem cells (hESC) and not in the fibroblast

cultures that are undergoing direct conversion into NPCs.

Figure 3. Immunocytochemical analysis with multiple markers demonstrate highly pure

preparations of iNPCs. At passage 20, the majority of cells in the culture were highly positive

for all NPC markers tested. Almost 100% cells were NESTIN-positive, and over 80% of cells

were positive for SOX1, PAX6, and PLZF, which are definitive neural markers.

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Figure 4. Examination of regional identity of iNPCs using quantitative gene expression

analysis shows dorsal-hindbrain identity. BJ and Rett syndrome (RTT) iNPC lines were tested.

The iNPC line derivation is summarized in Table 1.

Figure 5. Single-step direct conversion of mature neurons from hPSCs. (A) Schematic

presentation of neuron generation from hPSC by forced expression of Ngn2. Lentiviral vector is

designed to express an Ngn2-eGFP-puromycin resistant gene as a fusion protein.

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Figure 6. Quantitative analysis of iNPC-derived neurons differentiated by a single-step

protocol demonstrates highly pure neuron production. hNA identifies human cells while

MAP2 and NeuN mark dendrites and nuclei of neurons respectively.

Figure 7. Immunocytochemical analysis with multiple markers demonstrates tripotency of

iNPCs (the ability to generate all three neural lineage cells). MAP2 stains neurons, GFAP

stains Astrocytes, and O4 stains oligodendrocytes which appear later at 11 weeks.

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Figure 8. Generation and characterization of iNPCs from healthy adult fibroblasts. NPC

markers are evident (SOX2, SOX 1, PLZF, PAX6 and NESTIN) and the cells are proliferative

(Ki67). NANOG is not expressed showing that the cells are not pluripotent, and the GFP

retrovirus has been silenced in the iNPCs as expected.

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Figure 9. Generation and characterization of iNPCs from RTT patient derived fibroblasts.

Analysis was performed as in Figure 6.

Figure 10. Quantitative gene expression analysis showing the RTT-iNPC lines do not

express WT MECP2 transcript (red arrows).