Francis Deng*1, Siniša Hrvatin*

1, Philip diIorio

2, Alireza Rezania

3, Douglas Melton

1

1Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA

2Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 3BetaLogics Venture, Janssen Research and Development, LLC, Raritan, NJ

Abstract

High quality RNA isolation and transcriptome profiling of human pancreatic

cells purified by FACS using intracellular markers without reporters

Comparing hESC-derived,

fetal, and adult β-cells

Acknowledgments

We thank Anastasie Kweudjeu and Eliza Scadden for excellent

technical assistance.

Culture products of stem cell differentiation and somatic

cell reprogramming, as well as native tissues, exhibit high cell

type heterogeneity. Specific cell surface markers are often

unknown, hampering molecular analysis of defined

subpopulations. We report a method to obtain high-quality, global

gene expression profiles of primary human and hESC-derived cells

purified by fluorescence-activated cell sorting (FACS) using

intracellular/cytoplasmic markers.

The method enabled, for the first time, the discovery of the

molecular signatures of primary human fetal and adult pancreatic

beta-cells FACS-purified from other pancreatic cell types. These

included novel and known beta-cell factors. For instance, both

fetal and adult beta-cells were enriched for the transcription

factors Pdx1 and Nkx6-1, while only fetal beta-cells were enriched

for the earlier developmental regulators FoxA2 and Pax4, and

adult beta-cells preferentially expressed the maturity markers

MafA and Ucn3.

Subsequently, we compared these beta-cell transcriptomes

to that of insulin-positive cells derived from genetically

unmodified hESCs. Human ESC-derived insulin-positive cells were

more similar to fetal beta-cells than to adult β-cells, but also

lacked critical insulin processing/secretion machinery and

expressed many genes characteristic of other endodermal cell

types, Additionally, insulin-positive cells derived from multiple

pluripotent stem cell lines were highly similar.

We suggest that probing the differences in genes and

pathways between hESC-derived, fetal, and adult beta-cells will

provide heuristics for improving directed differentiation protocols

and generating functionally mature beta-cells that could

ameliorate type 1 diabetes upon transplantation. More generally,

this approach holds broad potential for comparing specifically

defined cell populations from in vitro stem cell-derived cultures

and native human tissues.

Modified intracellular FACS

Background

Overlaps between genes with enriched expression

(compared to non-β pancreatic cells) in human insulin+

cells derived from different sources. Noteworthy genes

and statistically significant gene ontology term

enrichment for each set are shown.

hESC-derived insulin+ cells share more enriched genes

with fetal β-cells than with adult β-cells. hESC-derived

insulin+ cells are missing or expressing low levels of key

β-cell transcriptional regulators, and aberrantly express

genes characteristic of other cell types (e.g. liver,

enteroendocrine, α-cells).

Hierarchical clustering of samples using probes that

are significantly differentially expressed between any

two cell types.

hESC-derived insulin+ cells cluster more closely with

fetal β-cells than with adult β-cells.

1. Modified intracellular FACS is a highly versatile approach that

permits isolating high-quality RNA from defined cell

populations without resorting to genomic modification

(reporters) or surrogate markers (surface antigens)

2. Insulin+ cells differentiated under similar conditions from two

hESC lines, H1 and HUES 8, are virtually indistinguishable.

3. On a global transcriptional level, hESC-derived insulin+ cells

resemble fetal β-cells more than mature β-cells.

Conclusions

In group-wise comparisons between H1- and HUES 8-

derived stage 6 insulin+ cells, only 328 probes (0.7%)

were significantly differentially expressed. No gene

ontology terms were significantly enriched in this set.

Insulin+ cells derived from

different hESC lines

Based on all detectably expressed genes, H1- and HUES

8-derived insulin+ cell samples cluster closely, often

closer than do cadaveric β-cells from separate donors.

Glucose stimulated insulin secretion

hESC-derived islet0

1

2

3

4

5

6 Low glucose

High glucose

KCl

***

******

pg

in

su

lin

secre

ted

per

cell

INS/

GC

G/D

NA

hESC-derived stage 6 differentiation

Intact adult human islet

Though directed

differentiation mimics

stages of

development, end-

stage in vitro cultures

are dysfunctional and

highly heterogeneous.

• To elucidate the basis of functional impairment and identify

targets for improving directed differentiation, it is important to

compare the transcriptomic profiles hESC-derived cells and

human in vivo-derived β-cells.

• Due to tissue/culture heterogeneity that can obscure salient

signals in one uncommon cell type, one must somehow

purify/isolate the insulin+ cells of interest.

• Cell surface markers and reporter lines face major technical

challenges for this purpose.

stage 6 culture

Fixed, permeabilized, stained Average signal; n=3

Illumina microarray on hESC-derived S6 cells

Fre

sh

Ave

rage

sig

nal

; n

=3

r2 = 0.9882 Best-fit: Y=0.01+1.0X

Standard staining protocol

A

B

Fres

h

Fixe

d/

Perm

’d

Stai

ned

RIN: 7.7

RIN: 5.9

RIN: 2.2

RIN: 5.4

RIN: 4.7

+RNasin Plus

RIN: 3.9 RIN: 8.4

C

FAC

S so

rted

Standard RNA isolation protocol

Protease digestion for 3 h at 50°C

5.6 ng/μl 6.0 ng/μl

Yield Quality0

5

10

15

20

25

0

2

4

6

8

10

pg

to

tal R

NA

per

cell

RN

A In

teg

rity N

um

ber

Optimized FACS

protocol preserves

RNA quality:

All initial steps at 4°C, with

concentrated RNasin Plus (RNase

inhibitor). Decontaminated and

DEPC-PBS-filled FACS sorter. No

high temp during RNA isolation.

Strategy

mRNA detection is unaffected by

the optimized protocol for

fixation/permeabilization,

intracytoplasmic staining, and

RNA isolation

insu

lin

glucagon

Human adult islets

hESC-derived stage 6 cells

glucagon and somatostatin

RNA integrity

assessed by

Bioanalyzer

Human fetal pancreas

empty

The modified FACS method can

distinguish populations defined

by intracellular hormones:

Separation/enrichment for cells

of interest was confirmed by

qRT-PCR for the hormone

genes insulin and glucagon.