SUPPORTING ONLINE MATERIAL

MATERIALS AND METHODS

Isolation of cells for gene expression profiling and culture

NCSCs were obtained by flow-cytometric isolation from timed pregnant Sprague-Dawley

rats (Charles River Laboratories, Wilmington, MA) as previously described (13). Briefly, E14.5

guts (stomach and intestines) were dissected into ice cold Ca, Mg-free HBSS (Gibco, Grand Island,

NY) and dissociated by incubating for 2 minutes at 37°C in 0.5mg/ml deoxyribonuclease type 1

(DNAse1, Sigma, St. Louis, MO; product D-4527). Cells were stained with antibodies against p75

(192Ig, directly conjugated to fluorescein) and α4 integrin (Becton-Dickinson, San Jose, CA; MR4-1

clone, directly conjugated to phycoerythrin), and NCSCs were isolated by flow-cytometry as

p75+α4+ cells (13, 14). Culture conditions for NCSCs were as described previously (14). To assess

the effect of GDNF on NCSC survival, the fraction of p75+α4+ cells that formed colonies after 6

days was compared in cultures containing 0, 1 or 10 ng/ml GDNF (R&D Systems, Minneapolis,

MN; product 512-gf). The number of cells per colony was also counted at 6 days to assess the effect

of GDNF on proliferation within NCSC colonies. The effect of GDNF on NCSC differentiation was

assessed by immunocytochemistry after culture for 14 days under standard conditions in the

presence of 0, 1, or 10 ng/ml GDNF. Cultures were fixed for 20 minutes at –20°C in 2.5% acetic

acid in ethanol then stained with antibodies against Peripherin (Chemicon, Temecula, CA) to

identify neurons, GFAP (Sigma) to identify glia, and SMA to identify myofibroblasts as previously

described (14).

In some experiments guts were dissected from E13.5-E15.5 mice derived from Ret+/- x

Ret+/- crosses, divided into two segments containing either the esophagus or the remainder of the gut

from stomach to hindgut, and dissociated individually in 0.5 mg/ml DNase1 as described above.

Unfractionated gut cells were assessed for p75 expression by flow cytometry (rat anti-mouse p75,

Chemicon, product MAB357) or cultured under non-adherent conditions as neurospheres using a

modified version of our standard medium with a 62.5/37.5 mixture of DMEM and Neurobasal

Medium (Gibco) and 0.8 µg/ml fibronectin added to the culture medium (Biomedical Technologies,

Stoughton, MA). Neurospheres were transferred to adherent cultures (plates were coated with PDL

and fibronectin as previously described (14)) to promote differentiation in order to assess

multipotency.

Gut neural crest migration assay

Guts were dissected from E13.5-E14.5 rat fetuses and embedded in collagen gels as

previously described (38) in the presence or absence of 10 ng/ml GDNF. Briefly, gels were made

by combining acidified collagen (BD Biosciences, Bedford, MA, product 354236), 1 M NaOH

(0.023 times the volume of collagen solution to be used), 5x OptiMEM medium (Gibco), and sterile

water according to the protocol provided by BD Biosciences to make a gel with a 1mg/ml final

collagen concentration. Each gut was embedded in 150µL of collagen solution. Beads (Cibacron

blue agarose, Sigma, product C1285) soaked in 10 µg/ml GDNF for at least one hour at 4°C were

used to assess directional migration of cells from the guts as previously described (28). After 2-3

days, guts were removed from the cultures and migrated cells were extracted by digesting the gels

with 0.05% trypsin (Gibco) and/or 1 mg/ml collagenase 4 (Worthington Biochemicals, Lakewood,

NJ) in HBSS without calcium and magnesium (Gibco) for 4 minutes at 37°C followed by quenching

on ice in 5 volumes of staining medium (14) containing 10% chick embryo extract. Isolated cells

were cultured for 14 days at clonal density then stained for the presence of neurons, glia, and

myofibroblasts to assess the potential of individual cells as described (6).

RNA amplification

Total RNA was extracted from 104 freshly isolated E14.5 gut NCSCs or E14.5 whole

fetuses using Trizol with 250µg/ml glycogen (Roche Diagnostic Corporation, Indianapolis IN).

RNA was extracted following the manufacturer’s instructions. 50ng of total RNA from each aliquot

of either the isolated NCSCs or whole fetus were used for RNA amplification. The extracted RNA

(30µl volume) was treated for 20min at 37°C with 2µl of RNase-free DNaseI (2U/µl; Ambion,

Austin TX) in the presence of 2µl of RNase inhibitor (10U/µl) (Invitrogen). The RNA was then

purified with RNeasy Mini Kit (Qiagen, Valencia CA) according to the manufacturer's instructions

and washed 3 times with 500µl of RNase-free water in a Microcon YM-100 (Millipore, Bedford

MA). After adding 0.025µg T7-d(T)24 primer (containing a T7 RNA polymerase binding sequence;

5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG(T)24,; Proligo, Boulder CO),

the RNA was dried down to 2.5µl. RNA was amplified through two consecutive rounds of

amplification using a modified version of the method of Baugh et al. (39). To make cDNA, first

strand was synthesized using T7-d(T)24 primer. After second strand synthesis, complementary RNA

(cRNA) was generated by T7 RNA polymerase (Promega, Madison WI). For the second round of

amplification, first strand cDNA was synthesized using random hexamers and second strand was

synthesized using the T7-d(T)24 primer. The double stranded cDNA was resuspended with 22µl

RNase-free water and transcribed to cRNA with the biotin labeling kit (BioArray Highyield RNA

transcript labeling kit (T7), Enzo Diagnostics, Farmingdale NY) for twelve hours. cRNA was

purified using the RNeasy Mini Kit. 60-80µg of biotinylated cRNA were obtained from two rounds

of RNA amplification from 50ng total RNA.

Hybridization and data analysis

After fragmentation (40), 15µg of NCSC or whole fetus cRNA were hybridized per chip to

Rat Genome U34 Arrays (Chips A, B and C; Affymetrix). The chips were hybridized and scanned

according to the manufacturer’s instructions. Signal intensities were read and analyzed using

methods described previously (41, 42). To measure fold changes, all negative signal intensity

values or values less than 100 were set to 100. To calculate the squared Pearson’s correlation

coefficient (R2) between two groups, we transformed each value to the base 10 logarithm (log10).

Log10 transformation is required because the Pearson’s correlation coefficient is designed to be

calculated based on normally distributed data, and the untransformed data are not normally

distributed (43). Although more statistically appropriate, the calculation of correlation coefficients

based on log10 transformed data causes these R2 values to be slightly lower than they would be if

calculated based on the untransformed data. The statistical significance of differences in signal

intensity for each probe set were evaluated by student's T-test using the log10 transformed values

from 3 independent replicates per cell type.

Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from gut NCSCs or whole embryo and treated with DNase1.

After purification of the RNA with the RNeasy mini kit (Qiagen), cDNA was made by reverse

transcription with 1µg random hexamer. The cDNA was extracted with phenol-chloroform and

precipitated with 20µg glycogen. After dissolving the cDNA with RNase-free water, cDNA

equivalent to 200 NCSCs was used for each PCR reaction. qRT-PCR was performed in triplicate

using three independent RNA samples. In the case of low-abundance templates that did not amplify

prior to 35 cycles of PCR, more cDNA was added until template amplification could be detected

before 35 cycles. Primers were designed to have a Tm of ~59°C and to generate short amplicons

(100-150bp). The PCR reactions were performed using a LightCycler (Roche Diagnostic

Corporation) according to the manufacturer's instructions. To control for the amplification of

genomic DNA, control reactions were performed after omitting reverse transcriptase from the cDNA

reaction. The RNA content of samples compared by qRT-PCR was normalized based on the

amplification of ß-actin. To estimate the magnitude of the difference in the expression levels of

individual RNAs between samples, we assumed that one cycle difference in the timing of

amplification by qRT-PCR was equivalent to a 1.8-fold difference in expression level (90%

amplification efficiency) (44).

SUPPLEMENTARY FIGURES

Supplementary Figure 1: Comparison of the expression of selected genes by qRT-PCR

between E14.5 gut p75+α4+ NCSCs and E14.5, E19.5, or P4 p75med restricted neural crest

progenitors/differentiated cells. Three independently isolated aliquots of each cell population

were sorted into Trizol, then RNA was extracted, random primed, and the expression of

individual genes was compared by qRT-PCR. Panel A presents results normalized based on cell

content (i.e. RNA from 200 cells of each population was compared). On this basis, each of the

Hirschsprung-associated genes (Ret, Sox10, Gfra1, and EDNRB) and most of the remaining 17

genes were expressed at significantly lower levels in the p75med cell populations (*, p

intensity are normalized between cell populations for microarray analysis. Even when RNA

content was normalized based on ß-actin, the Hirschsprung-associated genes and many of the

remaining 17 genes were expressed at significantly higher levels in the NCSCs as compared to

the p75med cell populations (*, p

only 5% of cells forming colonies that could be detected after 14 days in culture and 7.7±13.3%

of these colonies being multilineage. The remaining colonies contained neurons and glia

(7.3±7.1%), glia and myofibroblasts (11±11%), glia-only (31±11%), or myofibroblasts-only

(43±24%). Thus while the p75med populations contained some NCSCs, they were depleted of

NCSCs relative to the p75+ populations at each age.

Supplementary Figure 2: GDNF sometimes increased the density of cells within NCSC

colonies, but only at late time points in culture after differentiation started. E14.5 p75+α4+

cells isolated from the stomach/intestine were cultured at clonal density either in standard

medium (A,C,E,G) or in the presence of 10ng/ml GDNF (B,D,F,H). Panels A-D are tiled photos

of DAPI staining to reveal cell nuclei, while Panels E-H are tiled photos of brightfield images to

reveal staining with the neuronal marker Peripherin (black). After 6 days in culture NCSC

colonies contain mainly undifferentiated cells and most cells appear to be multipotent upon

subcloning (14). At this time in culture, colonies were indistinguishable whether they were

cultured in the absence (A) or presence of GDNF (B). After 14 days, some of the colonies

cultured in the presence of GDNF appeared larger (D), with more neurons (F) than were

observed in the NCSC colonies cultured in the absence of GDNF (C,E). Panels C and E

represent the same colony and panels D and F represent the same colony. Higher magnification

images of one field of view from the boxed region of the colonies show that the colony cultured

in GDNF (H) had neurons with much longer and thicker neurites than the colony cultured in the

absence of GDNF (G). This suggested that GDNF exerted a trophic effect that promoted the

differentiation or maturation of neurons. This is consistent with previous studies that observed

increased survival and neurogenesis of gut neural crest cells in the presence of GDNF (26, 27,

31).

Because this effect only became apparent after 11 days in culture when differentiation

was occurring within the colonies it suggested that the GDNF was promoting the survival or

proliferation of restricted neuronal progenitors that arose within stem cell colonies, rather than

the stem cells themselves. To confirm this we subcloned stem cell colonies cultured in the

presence or absence of GDNF for 11 to 15 days. There was no significant difference in the

number of multipotent daughter cells that were subcloned from individual NCSC colonies treated

(642±334) or untreated (524±407) with GDNF. This demonstrates that although some colonies

cultured in the presence of GDNF are larger and have more neurons, they do not contain

significantly more multipotent progenitors. Coupled with the fact that we could not detect any

effect of GDNF on the survival or proliferation of NCSCs during the first 7 days in culture, this

indicates that GDNF promotes the survival or proliferation of restricted progenitors under these

conditions.

Supplementary Figure 3: NCSCs from Ret-/- mice proliferate and differentiate normally in

culture. Cells isolated from E13.5 Ret+/+, Ret+/- and Ret-/- mouse guts formed neurospheres after 8

days in standard medium (A-C). Although NCSC frequency was significantly reduced in the Ret-/-

esophagus (Fig. 2), the neurospheres that did form did not differ significantly from those obtained

from normal littermates in size (D) or ability to form neurons when transferred to adherent cultures

(E, F). They also did not differ in terms of self-renewal potential as Ret+/+, Ret+/- and Ret-/-

neurospheres (cultured from E13.5 esophagus) that were subcloned to secondary cultures after 14

days in primary cultures formed 228±187, 308±236, and 280±225 secondary neurospheres

respectively (2 experiments totaling 14 to 34 neurospheres per treatment).

Supplementary Figure 4: qRT-PCR comparison of Ret expression by gut NCSCs and whole

fetus. (A) Fluorescence versus cycle number plot of ß-actin and Ret transcripts from gut NCSCs

and whole fetus by a LightCycler qRT-PCR assay based on SYBR Green dye detection. cDNA

equivalent to 200 cells was used for each PCR reaction. Although levels of ß-actin RNA were

equivalent in gut NCSC and whole fetus samples, Retamplified from NCSC samples after fewer

cycles than from whole fetus samples indicating that Ret RNA was present at higher levels in

NCSCs. For purposes of estimating the relative difference in Ret expression between samples

we assumed the efficiency of amplification was 90%, a typical value (44). The formula for fold-

change in Ret levels (FC) between gut NCSCs and whole fetus is FC=1.8x where x=(the number

of cycles to reach threshold (CT) of Ret of whole fetus - CT of ß-actin of whole fetus)-(CT of Ret

of gut NCSCs - CT of ß-actin of gut NCSCs). The cycle threshold (CT) for each gene was 18.71

(ß-actin of gut NCSCs), 18.82 (ß-actin of whole fetus), 22.74 (Ret of gut NCSCs) and 30.68 (Ret

of whole fetus). In these samples, gut NCSCs expressed 100-fold more Ret RNA than whole

fetus. The data summarized in Table 2 are based on comparisons of three independent samples of

gut NCSCs and whole fetal RNA. (B) A melting curve analysis indicated that only a single

product was amplified during the PCR reaction, and that the melting temperature of that product

matched the predicted melting temperature of the Ret amplicon (Tm=86.4°C). (C) qRT-PCR

products were separated in 2% agarose gels to confirm the presence of a single band of the

expected size (130bp). The PCR reaction was stopped at 22 cycles for ß-actin and 26 cycles for

Ret. Ret amplification was undetectable from whole fetus before 26 cycles.

Supplementary Figure 5: GDNF does not promote the survival or proliferation of E12.5 or

E14.5 p75+α4+ NCSCs in culture, even in chemically defined standard medium lacking

chick embryo extract. The presence of chick embryo extract in the culture medium had only a

modest effect on the survival and proliferation of gut NCSCs. Therefore we also tested the effect

of GDNF on NCSC survival and proliferation in chemically defined standard medium that

lacked chick embryo extract. Colonies from E12.5 p75+α4+ cells appeared indistinguishable after

6 days whether they were cultured in the presence (B) or absence (A) of 10ng/ml GDNF in

standard medium lacking chick embryo extract. GDNF did not promote the survival (C, E, G) or

proliferation (D, F, H) of E12.5 gut NCSCs cultured for 6 days in medium lacking chick embryo

extract (C, D), or E12.5 gut NCSCs cultured in standard medium (E, F), or E14.5 gut NCSCs

cultured in medium lacking chick embryo extract (G, H). Each panel represents 2 to 3

independent experiments.

SUPPLEMENTARY TABLES

Supplementary Table 1: Microarray comparison of E14.5 uncultured gut NCSCs to E14.5

whole fetus RNA. See the Excel spreadsheet on the zip disk sent by mail containing all of the data

from the microarray comparison of E14.5 whole fetus RNA and E14.5 gut NCSCs.

Supplementary Table 2: Summary of transcripts that were expressed at higher levels in gut

NCSCs as compared to whole fetus RNA. See the Excel spreadsheet listing the microarray

results for all genes that were expressed at 3-fold higher levels in gut NCSCs and for which the

difference in expression level was statistically significant (p

Supplementary Table 3: A comparison of genes upregulated in gut NCSCs with genes

previously identified by Ramalho-Santos et al. (6) and Ivanova et al. (7) as being associated

with stem cell identity. Rat orthologs had to exhibit at least 75% sequence identity to the

corresponding mouse “stem cell gene”, the probe set corresponding to the rat ortholog on the

oligonucleotide array had to exhibit at least 75% identity to the mouse “stem cell gene”, and the

NCBI e (expect) value generated by blasting the rat and mouse sequences had to be less than e-

50.

Ramalho-

Santos et al.

Ivanova et al.

Previously identified “stem cell genes” 217 280

orthologs of “stem cell genes” on the rat arrays 167 155

Rat “stem cell genes” expressed by whole fetus 149 117

Rat “stem cell genes” expressed by NCSCs 149 111

Rat “stem cell genes” upregulated in NCSCs (FC>1.5, p1.5, p3, p3, p

SUPPLEMENTARY REFERENCES

38. D. Natarajan, M. Grigoriou, C. V. Marcos-Gutierrez, C. Atkins, V. Pachnis, Development

126, 157-168 (1999).

39. L. R. Baugh, A. A. Hill, E. L. Brown, C. P. Hunter, Nucleic Acids Res 29, E29. (2001).

40. M. Mahadevappa, J. A. Warrington, Nat Biotechnol 17, 1134-6. (1999).

41. D. S. Rickman et al., Cancer Res 61, 6885-91. (2001).

42. T. J. Giordano et al., Am J Pathol 159, 1231-8. (2001).

43. S. A. Glantz, Primer of Biostatistics (McGraw Hill, New York, ed. Third, 1992).

44. L. Fink et al., Nature Medicine 4, 1329-1333 (1998).

A

B

C

200bp

100bp

NCSC fetus NCSC fetus

ß-actin Ret

Tm

Gut NCSCs ß-actinWhole fetus ß-actinGut NCSCs RetWhole fetus Ret

Gut NCSCs RetWhole fetus Ret

Supplementary figure 4: Iwashita et al.