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Biotechnology Letters ISSN 0141-5492 Biotechnol LettDOI 10.1007/s10529-014-1662-7

Generation of islet-like cell aggregates fromhuman non-pancreatic cancer cell lines

Mohammad Mahboob Kanafi, MuraliKrishna Mamidi, Shalini KashipathiSureshbabu, Pradnya Shahani,Chandravanshi Bhawna, et al.

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ORIGINAL RESEARCH PAPER

Generation of islet-like cell aggregatesfrom human non-pancreatic cancer cell lines

Mohammad Mahboob Kanafi • Murali Krishna Mamidi •

Shalini Kashipathi Sureshbabu • Pradnya Shahani •

Chandravanshi Bhawna • Sudha R. Warrier • Ramesh Bhonde

Received: 8 March 2014 / Accepted: 3 September 2014

� Springer Science+Business Media Dordrecht 2014

Abstract To explore a novel source for the deriva-

tion of islets, we examined the differentiation potential

of human non-pancreatic cancer cell lines, HeLa

(cervical carcinoma cell line) and MCF-7 (breast

cancer cell line). These cells were subjected to a

serum-free, three-step sequential differentiation pro-

tocol which gave two distinct cell populations: single

cells and cellular aggregates. Subsequent analysis

confirmed their identity as pancreatic acinar cells and

islet-like cell aggregates (ICAs), as evidenced by

amylase secretion and diphenylthiocarbazone staining

respectively. Reverse transcriptase-PCR and immu-

nocytochemistry assessment of the ICAs revealed the

expression of pancreatic specific markers Ngn-3, Glut-

2, Pax-6 and Isl-1. These ICAs secreted insulin in

response to glucose challenge, confirming their

functionality. We propose that ICAs generated from

HeLa and MCF-7 cell lines could form a promising

in vitro platform of human islet equivalents (hIEQs)

for diabetes research.

Keywords Breast cancer cell (MCF-7) �Differentiation �HeLa cells �Human islet equivalents �Islet-like cell aggregates

Introduction

The worldwide increase in the prevalence of diabetes

mellitus over last three decades has lent an urgency to

search for therapeutic solutions (Perez-Armendariz

2013). Pancreatic islet transplantation is a new

approach for the treatment of type-I diabetes. Inade-

quate supply of cadaveric islet cells and the lack of

new sources of glucose-responsive, insulin-producing

cells have hampered islet transplantation programs

(Ouyang et al. 2014). This scenario is further com-

pounded by the shortage of pancreatic donors thus

encouraging the search for non-conventional sources

of surrogate beta cells (Mfopou and Bouwens 2013;

Bhonde et al. 2014) for therapeutic purposes and

diabetes research. This is particularly important as

non-primate mammalian islets do not necessarily

reflect human islet pathophysiology (Bhonde et al.

1995). Thus, there is a pressing need to look for

alternative sources of human islet equivalents (hIEQ).

Mohammad Mahboob Kanafi and Murali Krishna Mamidi have

contributed equally to this work.

Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-014-1662-7) contains supple-mentary material, which is available to authorized users.

M. M. Kanafi � M. K. Mamidi � S. K. Sureshbabu �P. Shahani � C. Bhawna � S. R. Warrier � R. Bhonde (&)

School of Regenerative Medicine, Manipal University,

Bangalore 560065, India

e-mail: rr.bhonde@manipal.edu

S. R. Warrier

School of Biomedical Sciences, Faculty of Health

Sciences, Curtin University, Perth, WA 6845, Australia

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DOI 10.1007/s10529-014-1662-7

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PANC-1, a pancreatic adenocarcinoma cell line,

has been employed to demonstrate the generation of

islet-like cell aggregates (ICAs). FGF-2 stimulation,

after exposure to serum-free medium, has been shown

to induce clustering of PANC-1 cells into ICAs

(Hardikar et al. 2003). Increased expression of

pancreatic transcription factors, PDX-1 and PAX-6,

and endocrine cell markers, insulin and glucagon, has

been documented in PANC-1 cells (Wu et al. 2010).

Several other reports have also demonstrated the

generation of ICAs from PANC-1 cells by employing

different treatment regimen (Challa et al. 2011; Hiram

et al. 2012). However, there are no studies on

harnessing the potential of non-pancreatic cancer cell

lines to differentiate into islet cell lineage.

To identify easily available, abundant cell sources,

we examined the possibilities of using established, non-

pancreatic cancer cell lines to generate hIEQs. HeLa is

the oldest and most commonly studied human cancer

cell line derived from cervical cancer cells (Rahbari

et al. 2009), and MCF-7 is a widely used human breast

cancer cell line. In the present investigation, we

explored a pancreatic lineage differentiation potential

of HeLa and MCF-7 cell lines using a highly

reproducible and well established three step protocol.

Materials and methods

Culture of HeLa and MCF-7 cells

Human cancer cell lines, HeLa and MCF-7, were

cultured in knockout Dulbecco’s modified Eagle’s

medium (KO-DMEM) supplemented with 10 % (v/v)

fetal bovine serum (FBS), 5 mM L-glutamine and 50

U penicillin/streptomycin/ml. Cells were maintained

up to confluency, trypsinized and induced for pancre-

atic lineage differentiation.

In vitro differentiation of HeLa and MCF-7 cells

into ICAs

HeLa and MCF-7 cells were trypsinized and 106 cells

were re-suspended in serum free medium-1 (SFM-1) and

plated on ultralow adherent tissue culture dishes to induce

differentiation. This was carried out in three stages using

SFM-1, 2 and 3 for 10 days. SFM-1 contained KO-

DMEM (invitrogen), 1 % (w/v) bovine serum albumin

(BSA) Cohn fraction V, fatty acid free, insulin-

transferrin/selenium (ITS), 4 nM activin A and 1 mM

sodium butyrate. On the 3rd day, the medium was

changed to SFM-2, which contained KO-DMEM, 1 %

(w/v) BSA, ITS and 0.3 mM taurine. On 5th day, cells

were shifted to SFM-3, which contained KO-DMEM,

1.5 % (w/v) BSA, ITS, 3 mM taurine, 100 nM glucagon-

like peptide-1 and 1 mM nicotinamide, and fed with the

same media every 2 days until the 10 days. (All chem-

icals from Sigma-Aldrich unless otherwise indicated).

Amylase test

Detection of amylase secreted by differentiated cells

was carried out using the starch/agar plate method:

2 % (w/v) starch and 1.5 % (w/v) agar was prepared

and plated on 90 mm plates. 10 ll supernatants of day

3 (SFM-1), day 5 (SFM-2) and day 10 (SFM-3) was

added to the plates and incubated at 37 �C for 1 h.

Starch digestion by amylase was confirmed using 1 %

(v/v) I2. Salivary amylase and water were used as

positive and negative controls respectively.

Characterization of ICAs using

diphenylthiocarbazone staining

Specificity of ICAs was determined by diphenylthioc-

arbazone (DTZ; Sigma-Aldrich) staining. ICAs were

incubated with 10 mg/ml DTZ stain, dissolved in

DMSO, for 1 h at 37� C and viewed under an inverted

phase contrast microscope.

Reverse transcriptase PCR

Total RNA extraction and complementary DNA

synthesis was carried out as per the protocol reported

by Kanafi et al. (2013). Gene expression profile of

ICAs collected on day 3, 5 and 10 was compared with

un-induced HeLa and MCF7. The primer sequences

used in this study are listed in Supplementary Table 1.

18 s RNA was used as a housekeeping gene control.

Immunocytochemistry

ICAs were fixed for 20 min in 4 % (w/v) paraformal-

dehyde in chamber slides and treated with 0.1 % (v/v)

Triton X-100 to permeabilize the cell membrane. Cells

were blocked at room temperature in 5 % (w/v) BSA

solution for 30 min and incubated overnight at 4 �C

with mouse anti-human antibodies to Ngn-3 (BD

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Biosciences), Isl-1 (Abcam), C-peptide (Abcam) and

Glut-2 (Abcam). Subsequently, cells were washed in

PBS and incubated with FITC-conjugated with anti-

mouse secondary antibodies at room temperature for

2 h. Slides were counterstained with 40,6-diamidino-2-

phenylindole (DAPI) for 2–3 min and fluorescent

images were captured (Nikon).

Insulin release assay

One hundred ICAs derived from HeLa and MCF-7

were incubated with Krebs–Ringer bicarbonate buffer

(pH 7.4) supplemented with 10 mM Krebs–Ringer

bicarbonate HEPES medium and 5.5 mM glucose for

1 h at 37 �C. Cell supernatant was collected and stored

at -80 �C. ICAs were then transferred to Krebs–

Ringer bicarbonate HEPES medium supplemented

with 16.5 mM glucose. After incubation for 1 h at

37 �C, the supernatant was collected and stored at -

80 �C. Secreted insulin was measured using human

insulin enzyme-linked immunosorbent assay kit

(Mercodia, AB, Sweden).

Results

In vitro differentiation of human cancer cell lines

HeLa and MCF-7 into pancreatic lineages

The HeLa and MCF-7 cancer cell lines proliferated as

adherent monolayers of epithelial cell morphology

(Fig. 1a, e) and aggregated into spherical cell clusters

upon trypsinization and subsequent exposure to SFM-1

(Fig. 1b, f). In the first stage, definitive endoderm

differentiation was induced with ITS, activin A and

sodium butyrate. Pancreatic endoderm was induced in

stage 2 (SFM-2; Fig. 1c, g) with 0.3 mM taurine, and

finally pancreatic hormone-expressing ICAs were

induced with GLP-1, nicotinamide and increased levels

of taurine (3 mM) for 5 days (SFM-3; Fig. 1d, h).

Fig. 1 Pancreatic islet cell differentiation: Generation of islet-like cell aggregates (ICAs) from HeLa (a–d) and MCF-7 (e–h) through a

three-step induction protocol (scale bar 100 lm)

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Confirmation of ICAs by DTZ staining

At day 10 ICAs from both HeLa and MCF-7 showed

positive staining for DTZ, a zinc-chelating agent,

known to selectively stain pancreatic b cells due to

their high zinc content (Fig. 2b, d). The single cells

observed during the differentiation process (Fig. 2a–

d) did not stain positive for DTZ. The number of ICAs

obtained from HeLa (450 ± 25) was higher when

compared to ICAs from MCF-7 (250 ± 25) cell line.

Amylase secretion mimics in vivo pancreas

development

The presence of amylase in the culture supernatant

collected on days 3, 5 and 10 was detected by starch

digestion assay. Maximum starch digestion, and thus

amylase activity, was on day 3 and the least on day 10

(Fig. 2e, f). This feature mimics the milestones of

pancreatic development in vivo, wherein the exocrine

pancreas develops before endocrine pancreas. This

data also strongly indicates that the source of amylase

was exocrine pancreatic cells.

Characterization of ICAs

We compared the gene expression profile of pancre-

atic markers at day 0 for un-induced HeLa and MCF-7

cells with those of ICAs collected on day 3, 5 and 10

after induction. A significant up-regulation of pancre-

atic-specific transcripts, namely, Ngn-3, Glut-2, Pax-6

and Isl-1 was observed in the ICAs (Fig. 3a), whereas

un-induced HeLa and MCF-7 cells were negative for

these markers. These results were further confirmed by

Fig. 2 Diphenylthiocarbazone (DTZ) staining and amylase

secretion: ICAs stained positive for DTZ staining from HeLa

derived (b) and MCF derived ICAs (d) and the respective

unstained pictures represented in a and c (scale bar 100 lm).

Amylase secretion decreased progressively from serum free

medium (SFM)-1 to 3 for both HeLa (e) and MCF (f). Water and

salivary amylase was used as negative and positive controls

respectively (first and last plates of panels e and f)

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immunocytochemical staining of day 10 mature ICAs

for pancreatic specific markers. As seen in Fig. 4,

ICAs derived from HeLa and MCF-7 showed the

expression of early pancreatic markers such as Ngn-3

(Fig. 4a–f) and Isl-1(Fig. 4g–l) by day 10. We also

observed that these ICAs expressed b-cell-specific

glucose transporter Glut-2 (Fig. 4m–r) along with C-

peptide (Fig. 4s–x), which is a true marker of insulin

producing cells as it is endogenously generated upon

the cleaving of pro-insulin. These results further

confirm the islet lineage differentiation of HeLa and

MCF-7.

Fig. 3 Gene expression

profile of HeLa and MCF-7

derived islet-like cell

aggregates (ICAs): RT-PCR

analysis of day 5 and day 10

ICAs showed enhanced

expression of pancreatic

markers Ngn-3, Glut-2, Pax-

6 and Isl-1 when compared

with day 3 ICAs. Un-

induced HeLa and MCF-7

did not show the expression

of these pancreatic specific

genes. 18 s RNA was used

as a housekeeping internal

control gene

Fig. 4 Immunocytochemistry of day 10 islet-like cell aggre-

gates (ICAs): Pancreatic specific markers Ngn-3 (HeLa, a–c;

MCF-7, d–f), Isl-1 (HeLa,g–i; MCF-7, j–l), Glut-2 (HeLa, m–o;

MCF-7, p–r) and C-peptide (HeLa, s–u; MCF-7, v–x) was

observed in day 10 ICA of both HeLa and MCF. DAPI was used

as a counter stain (scale bar 400 lm)

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Response of ICAs to glucose stimulation

Glucose stimulation of ICAs derived from HeLa and

MCF-7 demonstrated insulin secretion in response to

glucose stimulation. It was found to be 29 ± 6 mU/l

and 23 ± 5 mU/l at basal glucose level (5.5 mM

glucose) and 275 ± 18 mU/l and 165 ± 14 mU/l at

stimulated glucose level (16.5 mM glucose), respec-

tively (Fig. 5).

Discussion

We have demonstrated for the first time a pancreatic

lineage differentiation potential of non-pancreatic

cancer cell lines, HeLa and MCF-7, following a

stringent protocol. The spherical morphology of ICAs

derived from epithelial cells of HeLa and MCF-7 was

similar to those generated from mesenchymal stem

cells (MSCs) reported by us and other groups (Kadam

et al. 2010; Tsai et al. 2012). It is well known that zinc

is highly expressed in pancreatic beta cells. Our data

clearly demonstrate that the ICAs stained positively

for DTZ (Fig. 2b, d), a zinc-chelating agent exhibiting

islet specificity.

Although the differentiation of human pancreatic

adenocarcinoma cell line, PANC-1, into islet-like

aggregates has been reported (Hardikar et al. 2003;

Dubiel et al. 2012), here we show that non-pancreatic

HeLa and MCF-7 cell lines can be induced to express

pancreatic specific markers Pax-6, Isl-1, Ngn3, Glut2

and C-peptide. Amylase test revealed decreasing

levels of amylase after day 3 (Fig. 2e, f) with

minimum levels on day 10 (Fig. 2e, f). This decrease

in the amylase secretion could be attributed to the poor

survival of these single cells in serum-free medium.

Moreover, pancreatic acinar cells are highly fragile

and cannot be maintained for long periods in culture as

they lose their zymogen granules and functionality

(Kurup and Bhonde 2000). The appearance of amy-

lase-producing cells before the formation of ICAs

indicates the development of exocrine pancreas prior

to endocrine cells (Kim and Hebrok 2001). Moreover,

these ICAs exhibited insulin secretion in response to

glucose challenge indicating their functional status

(Fig. 5).

Although ICAs derived from the cancer cell lines

may not be suitable for transplantation purposes due to

their origin, these ICAs can be used as hIEQ for

screening hypoglycemic agents and insulin secreta-

gogues. The advantage of HeLa and MCF-7 over

PANC-1 is that these cell lines are easily available in

most of the cell culture laboratories. However, there

may not be a higher theoretical advantage of HeLa and

MCF-7 over PANC-1, except that these cell lines serve

as a suitable substitute for PANC-1 as revealed by our

data.

Conclusion

We demonstrate, for the first time, an easy and

economical way of pancreatic differentiation from two

well-known human cancer cell lines. These functional

ICAs generated from non-pancreatic cell lines are a

novel source of hIEQs for screening anti-diabetic

compounds, offering an alternate source to animal

experimentation. Another important feature of our

study is the easy accessibility and abundant availabil-

ity of human cancer cell lines for generating hIEQs on

Fig. 5 Response of islet-

like cell aggregates (ICAs)

to glucose stimulation:

Comparison of secreted

insulin from ICAs derived

from HeLa and MCF-7

demonstrated a pronounced

increase of insulin from

ICAs after glucose

stimulation with higher

response in HeLa derived

ICAs

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an industrial scale for high throughput screening.

However, it remains to be seen whether these ICAs are

normal or tumorigenic.

Acknowledgments The authors wish to acknowledge

Manipal University for continuous support. Thanks are also

due to Dr. TMA Pai endowment chair for financial support. The

authors acknowledge Dr Anjan Kumar Das for careful

proofreading of the manuscript.

Supporting Information Table 1—Primer sequences for

pancreatic specific genes.

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