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hESC- and hiPSC-derived Schwann cells are Molecularly Comparable and

Functionally Equivalent

Kathryn R. Moss, Ruifa Mi, Riki Kawaguchi, Jeffrey T. Ehmsen, Qiang Shi, Paula I.

Vargas, Bipasha Mukherjee-Clavin, Gabsang Lee, Ahmet Höke

PII:

S2589-0042(24)01077-0

DOI:

https://doi.org/10.1016/j.isci.2024.109855

Reference:

ISCI 109855

To appear in:

ISCIENCE

Received Date: 7 November 2022
Revised Date: 11 February 2024
Accepted Date: 26 April 2024

Please cite this article as: Moss, K.R., Mi, R., Kawaguchi, R., Ehmsen, J.T., Shi, Q., Vargas,

P.I., Mukherjee-Clavin, B., Lee, G., Höke, A., hESC- and hiPSC-derived Schwann cells are

Molecularly Comparable and Functionally Equivalent

ISCIENCE

(2024), doi:

https://doi.org/10.1016/

j.isci.2024.109855

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hESC- and hiPSC-derived Schwann cells are Molecularly

Comparable and Functionally Equivalent

Kathryn R. Moss

, Ruifa Mi

, Riki Kawaguchi

, Jeffrey T. Ehmsen

, Qiang Shi

, Paula I. Vargas

Bipasha Mukherjee-Clavin

, Gabsang Lee

1,3,4

, Ahmet Höke

1,3,

Department of Neurology, Neuromuscular Division; Johns Hopkins University School of Medicine; Baltimore,

MD, 21205; USA

Semel Institute for Neuroscience and Human Behavior; University of California, Los Angeles David Geffen

School of Medicine; Los Angeles, CA, 90095; USA

Department of Neuroscience; Johns Hopkins University School of Medicine; Baltimore, MD, 21205; USA

Institute for Cell Engineering; Johns Hopkins University School of Medicine; Baltimore, MD, 21205; USA

*Corresponding Author and Lead Contact:

Ahmet Höke MD, PhD
Johns Hopkins School of Medicine
855 N. Wolfe St.
Baltimore, MD 21205
Tel: 410-955-2227
Fax: 410-502-5459
Email: ahoke@jhmi.edu

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Summary

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve

injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating

development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered

the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human

induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains

controversial, we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived

Schwann cells generated with our previously reported protocol. We identified only modest transcriptome

differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally, both

cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration

animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol

are molecularly comparable and functionally equivalent.

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Introduction

Myelinating Schwann cell models are required to understand demyelinating diseases and axon

regeneration in the peripheral nervous system. Developing human models is ideal given that translating findings

from rodents to humans has proven difficult (i.e. ascorbic acid clinical trials for CMT1A)

. Schwann cells can

be harvested from human nerve biopsies, but this procedure is invasive and yields a finite quantity of cells.

Therefore, a more practical strategy is to generate human Schwann cells from pluripotent cells. Although

human embryonic stem cells (hESCs) are considered the gold-standard with respect to pluripotency, ethical

concerns over their use make human induced pluripotent stem cells (hiPSCs) a more attractive approach

2,3

However, the equivalence of hESCs and hiPSCs is a much-debated topic

. Therefore, it is essential to

determine if hESCs and hiPSCs differentiated with a particular protocol demonstrate molecular and functional

equivalence.

We have developed a differentiation protocol that generates sufficient quantities of Schwann cells that

can subsequently be purified by fluorescence activated cell sorting (FACS) for ɑ4-integrin CD49d (CD49d)

expression

. Here, we generated hESC- and hiPSC-derived Schwann cells using our established protocol and

performed transcriptomic, proteomic, and functional comparisons of these cells. Three established hESC lines

and three iPSC lines previously generated from healthy control dermal fibroblasts were used. The hESC and

hiPSC lines were efficiently differentiated into Schwann cells as demonstrated by expression of Schwann cell

markers by immunocytochemistry and RNA sequencing. Differential expression analysis of the RNA

sequencing dataset revealed only modest transcriptome differences and antibody array demonstrated

insignificant proteome differences. hESC- and hiPSC-derived Schwann cell function was evaluated in a chronic

denervation and regeneration animal model followed by behavioral, electrophysiological, and histological

assessment. Transplantation of hESC- and hiPSC-derived Schwann cells equally improved nerve regeneration

and function as compared to transplantation of heat killed hESC- and hiPSC-derived Schwann cells

demonstrating functional equivalence of these cells.

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Results

hESC and iPSC Schwann Cell Differentiation

Our previously established 21-day differentiation protocol was used to generate hESC- and hiPSC-

derived Schwann cells (Figure 1A)

. Three common hESC lines were used (H1 [male], H7 [female] and H9

[female]) and three iPSC lines previously generated from healthy control dermal fibroblasts were used

(GM01582 [11-year-old female donor], GM02623 [61-year-old female donor] and GM08398 [8-year-old male

donor]). CD49d-positive Schwann cells were purified by FACS

. Representative CD49d-positive cell counts

demonstrate a relatively high differentiation efficiency for both hESCs and hiPSCs (15-27%, Figure 1B-1C).

However, comparing the differentiation efficiency between hESCs and hiPSCs is difficult to interpret due to

multiple variables introduced by FACS sorting. Purified hESC- and hiPSC-derived Schwann cells were cultured

and immunocytochemistry was performed at passage two to evaluate cell morphology and expression of the

Schwann cell marker S100B (Figure 1D). Both hESC- and hiPSC-derived Schwann cells exhibit bi/tripolar

morphology akin to cultured primary Schwann cells and highly express S100B.

Modest Transcriptome Differences between hESC- and iPSC-derived Schwann Cells

RNA was isolated from passage two hESC- and hiPSC-derived Schwann cells and transcriptome

analysis was performed by RNA sequencing. One independent replicate for each stem cell line was included

with three total hESC lines (n=3) compared to three total hiPSC lines (n=3). mRNA levels for each gene were

averaged and compared between hESC- and hiPSC-derived Schwann cells. To confirm proper differentiation of

the hESCs and hiPSCs into Schwann cells, Fragments Per Kilobase Million (FPKM) values of Schwann cell

markers were analyzed. FPKM values for the integral Schwann cell/myelin genes

Dystonin

(

DST

, p=0.9193),

Myelin Basic Protein

(

MBP

, p=0.8058),

Peripheral Myelin Protein 22

(

PMP22

, p=0.8265),

Erb-B2 Receptor

Tyrosine Kinase 2

(

ERBB2

, p-0.9112) and

Nerve Growth Factor Receptor

(

NGFR

, p=0.7581) were averaged

and compared between hESC- and hiPSC-derived Schwann cells (Figure 2A). Comparing these average FPKM

values to the ubiquitous genes

Histone 1, H1b

(

HIST1H1B

, hESC FPKM: Mean = 259.00, SD =75.19 ; hiPSC

FPKM: Mean = 245.20, SD = 151.30) and

ATP Synthase F1 Subunit Alpha

(

ATP5A1

, hESC FPKM: Mean =

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32.30, SD = 3.65; hiPSC FPKM: Mean = 30.63, SD = 4.28) demonstrates relatively high expression of the

Schwann cell/myelin genes.

Differential expression analysis of our dataset identified 142 genes (FDR-adjusted p<0.05); 20 genes

demonstrated significantly higher mRNA levels in hESC-derived Schwann cells and 122 genes demonstrated

significantly higher mRNA levels in hiPSC-derived Schwann cells (Figure 2B, Table S2). 16,590 genes were

comparably expressed between hESC- and hiPSC-derived Schwann cells, including 46 integral Schwann cell

genes (Figure S1A, Table S1). Ingenuity Pathway Analysis was performed on the 142 significant genes to

identify significant canonical pathways (Figure 2C, Table S3). Both the significant genes and significant

canonical pathways were further investigated for links to Schwann cell/myelin function. A link was established

for the following canonical pathways: Estrogen Biosynthesis

, Methylglyoxal Degradation III

, CREB

Signaling in Neurons

, Melatonin Degradation II

9,10

, Retinol Biosynthesis

and Wnt/Ca+ Pathway

(Figure

2C). However,

Wnt Family Member 5A

(

WNT5A

, higher expression in iPSC-derived Schwann cells, FDR-

adjusted p=0.0289) is the only canonical pathway-implicated gene that is directly involved in Schwann cell

function, specifically proliferation

(Figure 2D, Tables S2-S3).

Interleukin 33

(

IL33

, higher expression in

iPSC-derived Schwann cells, FDR-adjusted p=0.0007) and potentially

Formyl Peptide Receptor 1

(

FPR1

higher expression in iPSC-derived Schwann cells, FDR-adjusted p=0.0001) are canonical pathway-implicated

genes that are involved in oligodendrocyte function suggesting they may also play a role in Schwann cells

13,14

(Figure 2D, Tables S2-S3). Of the remaining significant genes, only three have been demonstrated to have a

clear connection to Schwann cells:

Periostin

(

POSTN

, higher expression in iPSC-derived Schwann cells, FDR-

adjusted p=3.69x10

-8

Thioredoxin Interacting Protein

(

TXNIP

, higher expression in iPSC-derived Schwann

cells, FDR-adjusted p=0.0170) and

Brain Derived Neurotrophic Factor

(

BDNF

, higher expression in iPSC-

derived Schwann cells, FDR-adjusted p=0.0465) (Figure 2D, Table S2). POSTN and TXNIP are involved in

Schwann cell migration

15,16

whereas BDNF plays a role in Schwann cell proliferation, migration and

myelination

17-19

. Twelve additional genes are potentially involved in Schwann cell/myelin function and/or play

a role in oligodendrocytes (Figure 2D, Table S2).

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Given that many more genes demonstrate significantly increased mRNA levels in hiPSC-derived

Schwann cells (122 increased in hiPSC, 20 increased in hESC), we also performed GO enrichment analysis on

both sets of genes to help interpret this result

20-22

. There was no significant enrichment for biological processes

or molecular functions with the genes demonstrating increased mRNA levels in hESC-derived Schwann cells.

However, there was significant enrichment for both biological processes and molecular functions with the genes

demonstrating increased mRNA levels in hiPSC-derived Schwann cells. The significantly enriched biological

processes are all generally related to development and the only two involving the nervous system are Cranial

Nerve Morphogenesis (GO:0021602, FDR-adjusted p=0.0356) and Rhombomere Development (GO:0021546,

FDR-adjusted p=0.0421) (Table S4). The significantly enriched molecular functions relate to DNA binding and

transcription (Table S5). Taken together, our results indicate that there are minimal transcriptome differences

between hESC- and hiPSC-derived Schwann cells and most of the significantly differentially expressed genes

do not to have a bona fide or potential link to Schwann cell/myelin function.

We also evaluated the robustness of our differentiation protocol and confirmed the Schwann cell

identity of our cells by generating Schwann cells from one hESC line (H9) and one hiPSC line (GM02623) and

performing a second set of RNA sequencing with passage two cells using three independent replicates from

each. FPKM values for Schwann cell markers were then compared between hESC- and hiPSC-derived Schwann

cells and between RNA sequencing sets one and two. FPKM values from published datasets with rat Schwann

cells were also included as a reference (data accessible at NCBI GEO database

, accession numbers

GSE211336

and GSE177037

). Comparing the FPKM values for

DST

MBP

PMP22

ERBB2

NGFR

AHNAK Nucleoprotein

(

AHNAK

S100 Calcium Binding Protein A6

(

S100A6

) and

S100 Calcium Binding

Protein A10

(

S100A10

) from RNA sequencing sets one and two demonstrates that our hESC- and hiPSC-

derived Schwann cells express Schwann cell markers although generally at lower levels than primary rodent

Schwann cells suggesting our cells are immature Schwann cells (Figure S1B, Table S6). And although there is

some variability, there is no statistically significant difference between hESC- and hiPSC-derived Schwann

cells or between sets one and two (Figure S1B). These results demonstrate the reproducibility of our

differentiation protocol to generate Schwann cells.

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Insignificant Proteome Differences between hESC- and iPSC-derived Schwann Cells

Cell lysates were collected from passage three hESC- and hiPSC-derived Schwann cells and proteome

analysis was performed by antibody array. One independent replicate for each stem cell line was included with

three total hESC lines (n=3) compared to three total hiPSC lines (n=3). Lysates were biotinylated and incubated

with two membranes containing a total of 1000 antibodies recognizing human proteins. Relative protein levels

were determined by immunoblotting for HRP-conjugated Streptavidin. Signal intensity for each protein was

averaged and compared between hESC- and hiPSC-derived Schwann cells. This analysis identified zero

proteins that were significantly differentially expressed (p<0.05, Figure 3A-3B, Table S7). Interestingly, pro-

BDNF (p=0.6219), BDNF (p=0.7774), IL33 (p=0.6219), five canonical pathway-implicated genes (none of

which are directly linked to Schwann cell function: CCL26, CCL28, MMP1, SAA1, IL24; p=0.7774, 0.9260,

0.7774, 0.6917, 0.5275, respectively) and SLPI (one of the twelve genes potentially linked to Schwann cell

function, p=0.4471) protein levels were included in the antibody array. These results suggest that some of the

significantly differentially expressed genes identified by RNA sequencing do not exhibit altered protein

expression. Although several proteins demonstrated a large difference between hESC- and hiPSC-derived

Schwann cells, none were significant due to high variability likely attributed to human genetic complexity

(Figure 3B, Table S7). The proteins that were most divergently expressed and nearest to statistical significance

are graphed in Figure 3C-3D. Given the large variability, the 18 most divergent expressed proteins and 4

proteins nearest to statistical significance were assessed for equal variance by F-test analysis. Btk, FGFR1 and

BNP are the only proteins in this group with unequal variance (Figure 3C-3D). The antibody array data for all

proteins were also analyzed by unpaired t-test with Welch correction to account for potential unequal variance

but no significant differentially expressed proteins were identified (Table S8). And only a portion of most

divergently expressed and nearest to statistical significance proteins have been linked to Schwann cell/myelin

function: Fibronectin (p=0.4089), Fibrinogen (p=0.3715) and Integrin alpha V (p=0.5132) for extracellular

matrix

26-28

, FGFR1 (p=0.4121) for differentiation and migration

29,30

, BNP (p=0.3772) for proliferation

and

Progranulin (p=0.1625) and MIF (p=0.1303) for nerve regeneration

32,33

(Figure 3C-3D).

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Functional Equivalence of hESC- and iPSC-derived Schwann Cells

Given that hESC- and hiPSC-derived Schwann cells are molecularly comparable, the functional

equivalence was evaluated. A chronic denervation and regeneration mouse model was used to determine the

ability of hESC- and hiPSC-derived Schwann cells to enhance peripheral nerve regeneration

in vivo

. Alive

and heat killed hESC- and hiPSC-derived Schwann cells were transplanted and regeneration of the transected

peroneal nerve into the denervated tibial nerve was evaluated histologically, electrophysiologically and

behaviorally. The average number of myelinated axons regenerated 5-7mm distal to the peroneal-tibial nerve

repair site was equivalent between alive hESC- and hiPSC-derived Schwann cells (p=0.9939, Figure 4A-4B).

Regenerated myelinated axons were also equivalent between heat killed hESC- and hiPSC-derived Schwann

cells (p=0.8341) but were significantly increased with alive hESC- and hiPSC-derived Schwann cell

transplantation as compared to heat killed (p=0.0005, Figure 4A-4B). Sciatic nerve compound muscle action

potentials (CMAPs) recorded from the distal foot muscles exhibited comparable amplitudes and latencies with

alive hESC- and hiPSC-derived Schwann cells (amp. p=0.8458, lat. p=0.1988, Figure 4C-4E). Again, CMAP

amplitudes and latencies were comparable between heat killed hESC- and hiPSC-derived Schwann cells (amp.

p=0.9606, lat. P=0.7916) but amplitudes were significantly increased (p=0.0033) and latencies were

significantly reduced (p=0.0092) with alive hESC- and hiPSC-derived Schwann cell transplantation as

compared to heat killed (Figure 4C-4E). A skilled walking behavioral test was used to quantify locomotion as a

readout of nerve and muscle function

. Given that Schwann cells derived from all hESC and hiPSC lines

comparably improved myelinated axon number and nerve electrophysiology, transplantation was performed

with Schwann cells derived from only one hESC and one hiPSC line. H9 (hESC) and GM01582 (hiPSC) were

randomly selected for this assay. After training the mice to walk on a ladder beam, their ability to grasp each

rung was subsequently scored as described using an established protocol

. The resulting injured hindlimb

scores were equivalent between alive hESC- and hiPSC-derived Schwann cells (p=0.0783, Figure 4F). These

findings suggest that hESC- and hiPSC-derived Schwann cells promote axon regeneration and reinnervation of

denervated distal foot muscles to a similar extent.

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Discussion

The field of stem cell differentiation is progressing at a rapid pace. Optimized protocols that improve

differentiation efficiency and efficacy are continuously being developed. Schwann cell differentiation protocols

are following this trajectory and have gone through several iterations

36,37

. We have developed a Schwann cell

differentiation protocol that is efficient and reproducible

. The resulting cells exhibit molecular, morphological,

and functional characteristics of Schwann cells

. Even though the equivalence of hESCs and hiPSCs is

controversial, reports comparing cells differentiated from each are scarce. However, in genetically matched

hESC and hiPSC lines, transcriptomic variation arising from genetic background overshadows variation due to

cellular origin

. Here, we sought to determine if hESC- and hiPSC-derived Schwann cells generated with our

protocol are molecularly and functionally equivalent in order to justify the sole use of iPSCs for future studies.

hESCs and hiPSCs were similarly differentiated with our protocol and the resulting hESC- and hiPSC-

derived Schwann cells were molecularly comparable. Only 0.85% of genes (142 out of 16,590) detected by

RNA sequencing were significantly differentially expressed and only a few of these had a concrete (2.82%, 4

out of 142) or potential (9.86%, 14 out of 142) link to Schwann cell/myelin function.

BDNF

WNT5A

POSTN

and

TXNIP

are the significantly differentially expressed genes at the mRNA level with an established role in

Schwann cells. Interestingly, 86% of the significantly differentially expressed genes exhibited increased

expression in iPSC-derived Schwann cells and these four genes belong to this group. GO enrichment analysis of

genes increased in iPSC-derived Schwann cells as compared to hESC-derived Schwann cells did not reveal

major links to nervous system function or provide much insight into the reason for this phenomenon. The

molecular similarity of hESC- and hiPSC-derived Schwann cells was also compared by antibody array. None of

the 1000 proteins quantified with this assay, including pro-BDNF and BDNF, were significantly differentially

expressed. Several additional integral Schwann cell/myelin proteins were also evaluated by the antibody array

including ERBB2 (p=0.7774), ERBB3 (p=0.7774), ERBB4 (p=0.7643), GDNF (p=0.9595), NGF (p=0.7644),

NGFR (p=0.3739), S100A4 (p=0.6219), S100A6 (p=0.4245) and S100A10 (p=0.3658). We cannot make

conclusions about why some genes are elevated at the mRNA level but not the protein level, but this is likely

due to complex post-transcriptional regulation that is involved in controlling the expression of these critically

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important genes. Our results indicate that hESC- and hiPSC-derived Schwann cells generated with our

differentiation protocol are molecularly comparable but identify genes that should be taken into consideration

for future experimentation and may contribute to line-to-line variability.

In support of our molecular analysis, we also demonstrated that transplanted hESC- and hiPSC-derived

Schwann cells function comparably to promote regeneration and reinnervation in a chronic denervation and

regeneration mouse model. It is well appreciated that some of the transplanted cells likely myelinated

regenerating axons, but the majority primarily promoted regeneration by providing trophic support

39,40

Myelinated axon number, CMAP amplitude and latency and skilled walking were all equally improved with

transplantation of alive hESC- and hiPSC-derived Schwann cells as compared to heat killed cells. These

findings further validate our Schwann cell differentiation protocol and provide evidence supporting the

molecular and functional equivalence of Schwann cells derived from hESCs and hiPSCs using this protocol.

Given the ethical concerns regarding the use of hESCs and the data we report here, we believe iPSCs are an

ideal platform for generating human Schwann cells, but it is best to be mindful of the minor differences that we

identified as compared to hESC-derived Schwann cells.

Future studies based on this work include exploring the myelination potential of hESC- and hiPSC-

derived Schwann cells to determine if gene expression changes in these cells become more evident with

maturation and affect myelin sheath function. Unfortunately, current Schwann cell differentiation protocols,

including our own, are either unable to myelinate or very inefficiently myelinate neurons

in vitro

5,37,41

. It may

be worth exploring functional differences in hESC- and hiPSC-derived Schwann cells caused by gene

expression changes like cell proliferation, migration and elongation. We would also like to note that

differentiation protocols requiring FACS sorting introduce multiple variables into the process of differentiation

Schwann cells. Although there are benefits to harvesting pure populations of Schwann cells, it would also be

advantageous to differentiate Schwann cells and neurons in a co-culture or organoid system in order to create

peripheral nerve-like environments with mature myelin sheaths. As Schwann cell differentiation protocols

continue to improve, protocols capable of producing pure cultured Schwann cells resembling primary cultured

Schwann cells and co-cultures with neurons ensheathed by mature myelin will likely become common practice.

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Limitations of the Study

Limitations of this study include not being able to compare CD49d-positive cells to CD49d-negative

cells to support the conversion to Schwann cells and not being able to analyze the subtypes of Schwann cells

differentiated from hESCs and hiPSCs. When developing the Schwann cell differentiation protocol

immunostaining prior to FACS sorting revealed that non-Schwann cell cells generated by this differentiation

protocol included Tuj1-positive neurons, presumably sensory neurons since they are neural crest derivatives.

We presumed the remaining cells were a combination of neural crest cells, melanocytes, other neural crest

derivatives and possibly connective tissue like fibroblasts, but we have not thoroughly characterized them due

to low survival after FACs sorting. Additionally, it would be interesting to define the subtype composition of

hESC- and hiPSC-derived Schwann cells which could be explored in future studies using single-cell RNA

sequencing.

Another

limitation of this study was only transplanting Schwann cells derived from one hESC and one

hiPSC line for the skilled walking assay to evaluate recovery in the chronic denervation and regeneration animal

model. However, Schwann cells derived from all three hESC and all three hiPSC lines were used to evaluate

recovery of tibial nerve myelinated fiber density, CMAP amplitude and CMAP latency in this model. The stem

cell lines used for the skilled walking assay were randomly selected. Given that hESC- and hiPSC-derived

Schwann cells are molecularly comparable and that hESC- and hiPSC-derived Schwann cells equally improved

tibial nerve myelinated fiber density, CMAP amplitude and CMAP latency, we elected to use only one hESC

and one hiPSC line for functional assessment given that these experiments are labor intensive and to conserve

mice.

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Acknowledgements

This work was supported by Maryland Stem cell Research Fund Discovery grant (2014-MSCRFI-0715)

and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation to Dr. Ahmet Hoke. Dr. Kathryn Moss

was supported by a Maryland Stem Cell Research Fund Postdoctoral Fellowship and is currently supported by

an NIH K22 Award (K22NS125057) and a Johns Hopkins Merkin Peripheral Neuropathy and Nerve

Regeneration Center Grant.

Author Contributions

Conceptualization, R.M., G.L. and A.H.; Methodology, B.M.-C., G.L.; Investigation, K.R.M., R.M.,

R.K., J.T.E., Q.S., P.I.V.; Formal Analysis, K.R.M., R.M., R.K.; Writing – Original Draft, K.R.M., A.H.

Writing – Review & Editing, K.R.M., A.H.; Visualization, K.R.M.; Supervision, G.L. and A.H.; Funding

Acquisition, A.H.

Declaration of Interests

The authors have declared no competing interests.

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Figure Titles and Legends

Figure 1. hESC and hiPSC cells are efficiently differentiated into Schwann cells.

(A) Diagram of the 21-

day Schwann cell differentiation protocol. (B) Representative cells count graphs of CD49d FACS sorted hESC-

and hiPSC-derived Schwann cells. (C) Representative Schwann cell differentiation efficiencies for each hESC

and hiPSC line (n=1 each). (D) Representative images of passage two CD49d-positive hESC- and hiPSC-

derived Schwann cells processed for immunocytochemistry with an anti-S100B antibody (red), Phalloidin to

label F-actin (green) and DAPI (blue). Scale bar, 50μm.

Figure 2. Minimal transcriptome differences between

hESC- and hiPSC-derived Schwann cells.

RNA was

isolated from passage two hESC- and hiPSC-derived Schwann cells and RNA sequencing was performed.

mRNA levels for each gene were averaged and compared between hESC- and hiPSC-derived Schwann cells

(n=1 per stem cell line, n=3 per stem cell type). (A) Average FPKM values of integral Schwann cell/myelin

genes. Data are represented as mean ± SEM. Equal variance was confirmed by F-test analysis. Unpaired t-test,

n.s. = not significant (p>0.05). (B) Differential expression analysis data displayed by volcano plot. Blue data

points are statistically significant (142 genes, FDR-adjusted p<0.05). (C) Statistically significant canonical

pathways identified by Ingenuity Pathway Analysis for the 142 significant genes. Light blue bars have

demonstrated links to Schwann cell/myelin function whereas white bars do not. (D) Significant differentially

expressed genes with concrete (light blue bars) and potential (light pink bars) links to Schwann cell/myelin

function. FDR-adjusted p-value are shown in parentheses.

Figure 3.

Insignificant proteome differences between

hESC- and hiPSC-derived Schwann cells.

Protein

lysates were collected from passage three hESC- and hiPSC-derived Schwann cells and antibody array was

performed. Signal intensity for each protein was averaged and compared between hESC- and hiPSC-derived

Schwann cells (n=1 per stem cell line, n=3 per stem cell type).

(

A) Representative immunoblots are shown. (B)

Differential expression results displayed by volcano plot. Data were analyzed by multiple unpaired t-tests with

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the false discovery rate approach two-stage step-up method of Benjamini, Kreiger and Yekutieli method

(desired FDR = 1%, significant p<0.05). The red line indicates statistical significance (p<0.05) with no data

points reaching significance. The gray boxes highlight proteins that were most divergently expressed (graphed

in (C)) and nearest to statistical significance (graphed in (D)). Average expression levels of (C) the most

divergently expressed proteins and (D) the proteins nearest to statistical significance. Data are represented as

mean ± SD. The p-values for those nearest to statistical significance are shown in parentheses. Proteins in red

have concrete links to Schwann cell/myelin function. Equal variance was assessed by F-test analysis and

proteins with unequal variance are outlined with a black box. The p-values from both unpaired t-tests (first p-

value) and unpaired t-tests with Welch correction (second p-value) are shown for these proteins in parentheses.

All data are not significant for both analyses (p>0.05).

Figure 4. hESC- and hiPSC-derived Schwann cells equally promote regeneration of chronically

denervated peripheral nerves.

Tibial nerves were chronically denervated, repaired with transected peroneal

nerve and heat killed or alive hESC- and hiPSC-derived Schwann cells were injected distal to the lesion.

Histological, electrophysiological, and behavioral recovery were evaluated three months post-transplantation.

(A)

Representative images of toluidine blue stained tibial nerve cross-sections with transplanted heat killed or

alive hESC- and hiPSC-derived Schwann cells. Scale bar, 10μm. (B) Quantification of tibial nerve myelinated

fiber number. (C) Representative sciatic nerve CMAP traces with transplanted heat killed or alive hESC- and

hiPSC-derived Schwann cells. Quantification of CMAP (D)

amplitude and (E)

latency. (F)

Skilled walking was

assessed by ladder beam test with transplanted alive hESC- and hiPSC-derived Schwann cells. Data are

represented as mean ± SEM. Heat killed hESC and hiPSC data were pooled (light pink bars) and compared to

pooled alive hESC and hiPSC data (light blue bars) to demonstrate improvement with alive cell transplantation.

Equal variance was assessed by F-test analysis and the pooled heat killed compared to pooled alive datasets all

demonstrated unequal variance (light pink/light blue bar graphs). The hESC- as compared to hiPSC-derived

Schwann cells datasets (black/white bar graphs) were analyzed by unpaired t-test due to equal variance and the

pooled heat killed compared to pooled alive datasets (light pink/light blue bar graphs) were compared by

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STAR Methods

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by

the lead contact, Ahmet Höke (ahoke@jhmi.edu).

Materials Availability

This study did not generate new unique reagents. Further information and requests for resources such as

reagents listed in key resources table should be directed to the lead contact.

Data and Code Availability

•

Both RNA-seq datasets have been deposited at GEO and are publicly available as of the date of

publication. Accession numbers are listed in the key resources table. Original antibody array blots have

been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in

the key resources table. Microscopy and nerve conduction data reported in this paper will be shared by

the lead contact upon request.

•

This paper does not report original code.

•

Any additional information required to reanalyze the data reported in this paper is available from the

lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Human Cells

Stem cell experiments were conducted with approval from the Johns Hopkins Institutional Stem Cell

Research Oversight Committee. The three human embryonic stem cell lines used in this study (H1 [male,

ethnicity/race unknown], H7 [female, ethnicity/race unknown] and H9 [female, ethnicity/race unknown]) were

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purchased from WiCell Research Institute. The three human induced pluripotent stem cells lines used in this

study (GM01582 [11-year-old Caucasian female donor], GM02623 [61-year-old Caucasian female donor] and

GM08398 [8-year-old Caucasian male donor]) were previously generated by Gabsang Lee from healthy control

dermal fibroblasts purchased from Coriell Institute for Medical Research. RRID for each are provided in the

key resources table. All stem cell lines were confirmed to have normal chromosome ploidy by karyotype

analysis; hESCs results are recorded in the Human Pluripotent Stem Cell Registry and hiPSCs were evaluated

after they were generated by Gabsang Lee. No apparent differences were detected due to biological sex, but our

sample sizes were likely insufficient to definitively conclude this.

All stem cell lines were cultured on Mitomycin C-treated CF6-Neo mouse embryonic fibroblast feeder

cells (MEFs, Thermo Fisher Scientific, A34964). Six-well plates were coated with 0.1% gelatin in water

(StemCell Technologies, 07903) and incubated at 37



C for 30 minutes prior to seeding approximately 300,000

MEFs per well in MEF medium (DMEM [Thermo Fisher Scientific, 11965092] supplemented with 10% FBS

[Thermo Fisher Scientific, 26140095]). MEFs were cultured in 5% CO

at 37º C overnight before seeding stem

cells. Thawed or passaged stem cells were seeded at approximately 20% confluency per well on MEF feeder

plates in stem cell medium (400ml DMEM/F-12 [Thermo Fisher Scientific, 11330032], 100ml knockout serum

replacement [Thermo Fisher Scientific, 10828028], 5ml 100x MEM non-essential amino acids solution

[Thermo Fisher Scientific, 11140050], 2.5ml 200mM L-glutamine [Thermo Fisher Scientific, 25030081], 500



2-mercaptoethanol [Thermo Fisher Scientific, 21985023] and 6ng/ml human basic fibroblast growth factor

[bFGF, Thermo Fisher Scientific, PHG0264, prepared in DPBS, no Calcium, no Magnesium {Thermo Fisher

Scientific, 14190144}]). Undifferentiated stem cells and differentiating/differentiated Schwann cells were

cultured in 5% CO

at 37º C in an incubator used solely for human stem cells which was routinely screened for

mycoplasma contamination. Medium was changed daily, and cells were passaged 1:3 using 5U/ml dispase

solution (StemCell Technologies, 07913) approximately every seven days after removing spontaneously

differentiated cells/colonies with a P200 pipet tip.

Mouse Strains

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Animal experiments were conducted with approval from the Johns Hopkins Animal Care and Use

Committee. Adult male NOD SCID mice (aged three-months) were obtained from the Jackson Laboratory.

RRID provided in the key resources table. Male mice were exclusively used given that no apparent biological

sex differences were observed in hESC- and hiPSC-derived Schwann cells and to minimize variability due to

the estrous cycle. NOD SCID mice are homozygous for the

Prkdc

scid

mutation resulting in the absence of

functional T cells and B cells, lymphopenia and hypogammaglobulinemia. They are able to accept allogeneic

and xenogeneic grafts making them an ideal model for cell transplantation experiments. Mice used for this

study were purchased directly from the Jackson Laboratory, so their backcrossing status, weight and husbandry

conditions were monitored and standardized though the Jackson Laboratory. Littermate males were shipped and

housed together in individually ventilated cages (Allentown Caging Equipment, PC75JHT) with corn cob

bedding (Teklad, 7097). Mice were provided a single cotton nestlet (Ancare Nestlets, NES3600) for enrichment,

fed autoclaved global 18% protein extruded rodent diet (Teklad, 2018SX) and provided reverse-osmosis-treated

water via an Edstrom automated in-cage watering system (Avidity Science LLC). Cages were changed every

two weeks and colony mice were monitored quarterly for pathogens. Mice were randomly assigned to groups

for Schwann cell transplantation experiments and differences due to biological sex were not evaluated due to

the sole use of male mice.

METHOD DETAILS

Schwann Cell Differentiation

MEF-conditioned stem cell medium was prepared by adding stem cell medium to MEF feeder plates,

collecting the medium after 24 hours and freezing at -20



C. Gelatin coated 10cm dishes (one per three wells of

stem cells) were prepared by incubating with 0.1% gelatin in water at 37



C for at least 30 minutes. Matrigel

coated 24-well plates were prepared by adding ice-cold LDEV-free, hESC-qualified matrigel (Corning Life

Sciences, 354277) to each well (thawed at 4



C overnight) and incubating at room temperature for at least one

hour. After removing spontaneously differentiated cells/colonies with a P200 pipet tip, cells were incubated

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with accutase (Innovative Cell Technologies, Inc., AT104) at 37



C for 20 minutes. Stem cell medium was

added to each well, the cells were gently triturated and then passed through a 40



m cell strainer (Corning Life

Sciences, 352340). The cells were topped up to 15ml with stem cell medium and centrifuged at 200xg for five

minutes at 20



C. Supernatant was removed, and cells were resuspended in MEF-conditioned stem cell medium

supplemented with 6ng/ul bFGF and 10



M Y-27632 (Cell Signaling Technology, 13624). Cells were seeded

onto matrigel coated 24-well plates, 60,000 cells per well, and medium was changed every two days until

reaching 80% confluency which generally takes one to three days.

The stem cells were then differentiated into Schwann cells following a protocol developed by the Lee

lab

. This involved treatment with small molecules in KSR medium (415ml knockout DMEM [Thermo Fisher

Scientific, 10829018], 75ml knockout serum replacement, 5ml 100x MEM non-essential amino acids solution,

2.5ml 200mM L-glutamine and 500



l 2-mercaptoethanol) and/or NB medium (480ml neurobasal medium

[Thermo Fisher Scientific, 21103049], 10ml B27 Supplement [Thermo Fisher Scientific, 17504044], 5ml N2

Supplement [Thermo Fisher Scientific, 17502048] and 2.5ml 200mM L-glutamine). On differentiation days

zero and one (cells at 80% confluency), all of the stem cell medium was gently removed by aspiration and 500ul

of KSR medium supplemented with 10μM SB-431542 (Tocris Bioscience, 1614) and 500nM LDN-193189

(Tocris Bioscience, 6053) was added to each well. Most of the medium was removed to prevent drying on all

subsequent medium changes (400



l removed per well, adding 500



l fresh medium each time). One

differentiation days two and three, the medium was changed to KSR medium supplemented with 10μM SB-

431542, 500nM LDN-193189, 3μM CHIR 99021 (Tocris Bioscience, 4423) and 10μM DAPT (Tocris

Bioscience, 2634). On differentiation days four and five, the medium was changed to KSR/NB (3:1) medium

supplemented with 3μM CHIR 99021 and 10μM DAPT. On differentiation days six and seven, the medium was

changed to KSR/NB (1:1) medium supplemented with 3μM CHIR 99021 and 10μM DAPT. On differentiation

days eight and nine, the medium was changed to KSR/NB (1:3) medium supplemented with 3μM CHIR 99021

and 10μM DAPT. On differentiation days 10 through 20, the medium was changed to NB medium

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supplemented with 200μM dibutyrl cyclic AMP (Sigma-Aldrich, D0627) and 200μM L-ascorbic acid (BioGems

International, Inc., 5088177).

Schwann Cell FACS Sorting and Culture

Matrigel coated 12-well plates were prepared by adding ice-cold matrigel to each well (thawed at 4



overnight) and incubating at room temperature for at least one hour. Poly-D-lysine (PDL, Sigma-Aldrich,

P7280, prepared in water) coated coverslips were prepared by placing autoclaved 12mm diameter #1.5

coverslips into a 24-well plate and dotting 100μl of 100μg/ml PDL onto each coverslip 50μl at a time. The plate

was incubated at 37



C for at least 30 minutes and then washed several times with PBS (Thermo Fisher

Scientific, 10010023). Schwann cells were purified by Fluorescence Activated Cell Sorting (FACS) sorting on

differentiation day 21. The medium was gently aspirated, and the cells were washed once with PBS. After

removing the PBS, ReLeSR (StemCell Technologies, 100-0483) was added to each well, incubated at room

temperature for one minute and removed by gentle aspiration. The dry plate was incubated at 37



C for three

minutes and then accutase was added to each well. The plate was tapped against a hard surface five times and

the cells were lifted by pipetting with P1000 pipet. The cells in accutase were transferred to a 15ml tube and

additional accutase was added up to 5ml. The tube was incubated in a 37



C water bath for 3 minutes with

gently agitation every minute. The cells were triturated in a clean 15ml tube 1ml at a time which generally

required 8-10 passes through the pipet tip. The dissociated cells were passed through a 40



m cell strainer and

MEF medium was added up to 15ml. The cells were centrifuged at 200xg for 10 minutes at 10



C. Supernatant

was removed, and cells were resuspended in 1ml FACS Buffer (40ml DPBS, no calcium, no magnesium, 10ml

MEF Medium and 100μl 10mg/ml DNaseI [Sigma-Aldrich, DN25]) supplemented with 10



M Y-27632. 50μl

of cells were transferred to a 1.5ml tube to use as unstained control and store on ice. The remaining cells were

transferred to a 1.5ml tube, 10μl PE-conjugated anti-CD49d antibody (R&D Systems, FAB1354P) was added

and the cells were rotated in the dark at 4



C for 30 minutes. The cells were transferred to a 15ml tube, 10ml

FACS Buffer supplemented with 10



M Y-27632 was added and the cells were centrifuged at 200xg for 5

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minutes at 10



C. Supernatant was removed, cells were resuspended in 10ml FACS Buffer supplemented with



M Y-27632 and the cells were centrifuged at 200xg for 5 minutes at 10



C. Supernatant was removed and

the cells were resuspended in FACS Buffer supplemented with 10



M Y-27632 at a concentration of

approximately five million cells/ml. The cells were transported on ice and sorted at the Johns Hopkins FACS

core facility. Singlet CD49d-positive Schwann cells were collected into a tube containing Schwann cell medium

(ScienCell Research Laboratories, 1701, 500ml basal medium, 25ml FBS, 5ml Schwann cell growth

supplement and 5ml antibiotic solution) supplemented with 10



M Y-27632. Schwann cells were seeded onto

matrigel coated 12-well plates, 70,000 cells per well, and PDL coated coverslips in 24-well plates, 35,000 cells

per well, in Schwann cell medium supplemented with 10



M Y-27632 (passage one). Medium was changed

every two days with Schwann cell medium without supplement. Schwann cells were passaged after reaching

80% confluency which typically required one week. Schwann cells were passaged 1:3 using 0.05% trypsin-

EDTA (Thermo Fisher Scientific, 25300054). Passage two hESC- and iPSC-derived Schwann cells were used

for RNA sequencing and immunocytochemistry. Passage three hESC- and iPSC-derived Schwann cells were

used for antibody array and transplantation.

Schwann Cell Immunocytochemistry

Passage two hESC- and iPSC-derived Schwann cells plated on PDL coated coverslips were fixed with

4% paraformaldehyde (Electron Microscopy Sciences, 15714) in PBS for 15 minutes at room temperature after

reaching 70-80% confluency. The paraformaldehyde was discarded, and the Schwann cell were washed with

PBS three times for 10 minutes each. The Schwann cells were permeabilized with 0.1% Triton X-100 (Sigma-

Aldrich, T9284) in PBS for 15 minutes with gentle agitation at room temperature and then washed with PBS

three times with gentle agitation for five minutes each. Schwann cells were blocked with 5% Normal Goat

Serum (Jackson ImmunoResearch Laboratories, Inc., 005-000-121) in PBS for one hour with gentle agitation at

room temperature and incubated with primary antibody (1:200 rabbit anti-S100B [Sigma-Aldrich,

HPA015768]) overnight with gentle agitation at 4



C. Schwann cells were washed with PBS three times with

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gentle agitation for 10 minutes each and then incubated with secondary antibody (1:400 Alexa Fluor 488

Phalloidin [Thermo Fisher Scientific, A12379] and 1:1000 goat anti-rabbit Alexa Fluor 568 [Thermo Fisher

Scientific, A11036]) for one hour with gentle agitation at room temperature. Schwann cells were washed with

PBS three times with gentle agitation for 10 minutes each and mounted onto slides with ProLong Gold Antifade

Mountant with DAPI (Thermo Fisher Scientifc, P36931). Z-stacks were acquired using a Zeiss Axio Imager Z1

with a 20X air objective using Zeiss Blue software and final images were prepared with Imaris (Oxford

Instruments).

RNA Isolation and RNA Sequencing

RNA was isolated from hESC- and iPSC-derived Schwann cells at passage two after reaching 90%

confluency with TRIzol (Thermo Fisher Scientific, 15596026) following the manufacturer’s protocol and

subsequently purified with a RNeasy Mini Kit (Qiagen, 74104). The Johns Hopkins Experimental and

Computational Genomics core facility evaluated RNA quality with a bioanalyzer, and libraries were prepared

using TruSeq Stranded RNA (100 ng, Illumina). Preparation included 100bp paired end reads and sequencing

run was carried out using an Illumina sequencing platform. Quality control (QC) was performed on base

qualities and nucleotide composition of sequences, to identify problems in library preparation or sequencing.

Sequence quality for the dataset described here was sufficient that no reads were trimmed or filtered before

input to the alignment stage. Reads were counted using HT-seq (version 2.0)

and aligned to the latest Human

reference genome (GRch38) using the STAR spliced read aligner (version 2.4.0)

. Average input read counts

were 66.7M per sample (range 54.8 to 76.30M) and average percentage of uniquely aligned reads was 80.6%

(range 72.3% to 88.7%). Low count transcripts were filtered, and count data were normalized using the method

of trimmed mean of M-values (TMM). Differentially expressed genes (FDR < 0.1) were then identified utilizing

the Bioconductor package EdgeR (version 2.7)

. In the first set, paired information was incorporated into the

model to account for pair-specific batch effects, while in the second set, paired information was omitted from

the model. FPKM values were based on the gene model using GRCh38 RefSeq downloaded from UCSC table

browser

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Antibody Array

Protein levels were quantified in passage three hESC- and iPSC-derived Schwann cells with the Human

L Series Array 1000 Membrane Kit (RayBiotech, AAH-BLM-1000-2). following the manufacturer’s protocol.

Briefly, cell lysates were biotinylated and incubated on membranes containing antibodies for 1000 human

proteins. Immunoblot with HRP-conjugated Streptavidin was then performed and the membranes were imaged.

Relative protein levels were quantified using the RayBio Analysis Tool and signal intensity of all samples were

normalized to the negative control value for GM01582 iPSC-derived Schwann cells. Signal intensity for each

protein was averaged and compared between hESC- and hiPSC-derived Schwann cells (n=1 per stem cell line,

n=3 per stem cell type) and compared by multiple unpaired t-tests (without and with Welch correction for

unequal variance) with the false discovery rate approach two-stage step-up method of Benjamini, Kreiger and

Yekutieli method in Prism 9 (desired FDR = 1%, significant p<0.05).

Literature Search for Schwann cell Function

A PubMed literature search was performed to investigate links to Schwann cell/myelin function on the

following: (1) 142 differentially expressed genes identified by RNA sequencing, (2) 20 significant canonical

pathways identified by Ingenuity Pathway Analysis, (3) 18 most divergently expressed genes identified by

antibody array and (4) four genes nearest to statistical significance identified by antibody array. The gene and

the term “Schwann cell,” “myelin” or “oligodendrocyte” were searched in PubMed. A concrete link to Schwann

cell function is defined by a research publication demonstrating a functional role for the gene in Schwann cells

whereas a potential link to Schwann cell function is defined by a research publication demonstrating a

functional role for the gene in oligodendrocytes or expression in Schwann cells without functional data.

Percentages were calculated based on the number of concrete and potential links divided by the total number of

genes assessed.

Schwann Cell Transplantation

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Three-month-old male NOD SCID mice were used for the established chronic denervation and

regeneration animal model

. This involved transecting the tibial nerve just distal to the sciatic trifurcation,

ligating both the proximal and distal stumps, and suturing distal stump to nearby muscles with 10-0 Sterile

Micro Sutures (AROSurgical Instruments Corporation, T05A10N10-13). The contralateral side was kept as an

uninjured control. Passage three hESC- and iPSC-derived Schwann cells were trypsinized (0.05% trypsin-

EDTA) and prepared at a concentration of 40,000 cells/μl in PBS. Heat killed cells were also prepared

by boiling at 100° C for 10 minutes, cooling on ice and boiling again at 100° C for 10 minutes. Four months

post-denervation (seven-month-old mice), the distal tibial nerve was transplanted with 2.5μl alive or heat killed

hESC- or iPSC-derived Schwann cells using a Hamilton syringe (model 75 RN syringe, Hamilton Company,

7634-01) and repaired with freshly transected proximal common peroneal nerve using 10-0 Sterile Micro

Sutures. The syringe was washed with PBS between cell transplantations. Three months post-repair (10-month-

old mice), regeneration was assessed by electrophysiological, histological and behavioral assays.

Nerve Electrophysiology

Motor

‐

evoked responses were evaluated using an established protocol

. Briefly, under isoflurane

anesthesia (flow rate 2 L/min), compound motor action potential (CMAP) in the distal foot muscles was

recorded from the injured and contralateral sciatic notch of each mouse upon stimulation with PowerLab

(ADInstruments) and subdermal needle electrodes (Natus Medical Store, 019-475900). Amplitude (peak to peak

from the strongest recorded response) and latency (delay from stimulation to response onset) were quantified

for each animal using Scope 4 and LabChart Reader (ADInstruments).

Nerve Morphometry

Immediately following electrophysiological examination, animals were sacrificed by cervical dislocation

and 2mm of t

ibial nerve was harvested 5mm distal to the suture site.

Nerve samples were fixed and processed

for light microscopy by preparing 0.5μm sections and staining them with 1% toluidine blue (Electron

Microscopy Sciences, 22050)

. Tiled images of the entire nerve cross section were acquired on a Zeiss

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Axiophot microscope with a 60x objective. The number and density of myelinated axons (healthy axons

surrounded by a myelin sheath) were evaluated from four randomly placed squares of equal area in the tibial

branch of the sciatic nerve with ImageJ

48,49

. Nerve electrophysiology and morphometry results were averaged

and compared between alive transplanted hESC- and iPSC-derived Schwann cells (n=2-4/stem cell line, n=8-12

total) and between heat killed transplanted hESC- and iPSC-derived Schwann cells (n=5-3 total) by unpaired t-

test. Transplanted hESC- and iPSC-derived Schwann cell results were also pooled and compared between alive

and heat killed cells by unpaired t-test with Welch correction for unequal variance (significant p<0.05, Prism 9).

Skilled Walking Behavior

Behavioral recovery was evaluated with a ladder beam walking task by training the mice at least three

times per week for two subsequent weeks and performing the test and analysis using an established protocol

Results were averaged and compared between transplanted H9 hESC-derived Schwann cells and GM01582

iPSC-derived Schwann cells (n=4 each) by unpaired t-test (significant p<0.05, Prism 9).

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were performed using Prism 9 which included the following tests: F-test for equal

variance, unpaired t-test (without and with Welch correction for unequal variance), multiple unpaired t-tests

with the false discovery rate approach two-stage step-up method of Benjamini, Kreiger and Yekutieli method

(without and with Welch correction for unequal variance) and one-way ANOVA with Tukey’s post-hoc. The

statistical test used, exact n, definition of what n represents and the definition of center and dispersion for each

analysis is located in the figure legends. Randomization was performed for the mouse transplantation studies by

randomly selecting mice for each condition from multiple litters. Sample size estimations were based on

previous experience with RNA sequencing and standard group sizes for rodent experiments. No data points

from any assay were excluded from this study.

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Excel-format Table Titles and Legends

Table S1. Differential expression analysis for all genes identified by RNA sequencing set one, Related to

Figure 2

Table S2. Differential expression analysis for significant genes identified by RNA sequencing set one,

Related to Figure 2

Table S3. Significant canonical pathways identified by Ingenuity Pathway Analysis of the significant

differentially expressed genes from RNA sequencing set one, Related to Figure 2

Table S6. FPKM values for RNA sequencing sets one and two, Related to Figure 2

Table S7.

Multiple unpaired t-test analysis of antibody array data, Related to Figure 3

Table S8.

Multiple unpaired t-test with Welch correction analysis of antibody array data, Related to

Figure 3

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KEY RESOURCES TABLE

REAGENT or
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SOURCE

IDENTIFIER

Antibodies

Human Integrin
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Qiagen

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Human L Series
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Deposited Data

RNAseq Datasets Gene Expression

Omnibus (GEO)

Accession Number: GSE208708

Antibody Array
Blots

Mendeley Data

DOI: 10.17632/k85ygcx787.2

Experimental Models: Cell Lines

H1 Human
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Software and Algorithms

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STAR spliced read
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2.4.0)

Bioconductor
package EdgeR
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Author: Mark
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McCarthy and
Gordon Smyth

https://www.bioconductor.org/packages//2.7/bioc/html/edgeR.html

UCSC Table
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Developed by
UCSC and
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Graphpad Prism 8
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GraphPad
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Zen2 Blue Edition
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Imaris x64
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