Biochemical and Biophysical Research Communications 418 (2012) 765–769

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate /ybbrc

New method to differentiate human peripheral blood monocytes into insulinproducing cells: Human hematosphere culture

Jin Hur a,b,1, Ji Min Yang a,c,1, Jae-Il Choi a,b,1, Ji-Yeon Yun a,b, Jae Hee Jang a,c, Joonoh Kim a,c,Ju-Young Kim a,b, Il-Young Oh a,c,d, Chang-Hwan Yoon a,d, Hyun-Jai Cho a,b, Young-Bae Park b,Hyo-Soo Kim a,b,c,⇑a National Research Laboratory for Stem Cell Niche, 101 Daehak-ro, JongRo-gu, Seoul, Republic of Koreab Innovative Research Institute for Cell Therapy, Seoul National University, 101 Daehak-ro, JongRo-gu, Seoul, Republic of Koreac Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Koread Cardiovascular Center, Seoul National University Bundang Hospital, 173 Beon gil, Bundang gu, Seongnam, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 January 2012Available online 28 January 2012

Keywords:Three-dimensional cultureInsulinInsulin producing cellsStem cellsSpheroidRegenerative medicine

0006-291X/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.bbrc.2012.01.096

Abbreviations: FBS, fetal bovine serum; DTZ, dithizmedia 2; DMSO, dimethyl sulfoxide; hPB, humanhuman peripheral blood mononuclear cells; Glc,hematosphere; NPCs, nestin-positive cells; IPCs, insul⇑ Corresponding author at: Department of Intern

University Hospital, 101 DaeHak-ro, JongRo-gu, SeouFax: +82 2 766 8904.

E-mail addresses: hyosoo@snu.ac.kr, gene44@hanm1 These authors contributed equally to this work.

Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one toget cell source of b-cell replacement therapy for diabetes. It has been suggested that islets and neuronsshare features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere(BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stagedifferentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cellsform islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on,such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2and PC2) or b cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone(DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived fromBBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novelprotocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy ofex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatmentof diabetes.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Diabetes mellitus (DM) has been widespread all around theworld affecting more than one hundred million patients, and it isexpected to increase dramatically in the next decade [1,2]. Type 1diabetes mellitus (T1DM) is an organ-specific autoimmune diseasein which insulin-secreting b-cells are irreversibly destroyed, result-ing in deficiency of circulating insulin [3]. To date administration ofexogenous insulin remains the main treatment in T1DM [1].However, the exogenous insulin injection is not physiologic andinevitably cause serious complications such as hypoglycemia. In

ll rights reserved.

one; EBM-2, endothelial basalperipheral blood; hPBMNCs,glucose; BBHS, blood-born

in-producing cells.al Medicine, Seoul Nationall 110-744, Republic of Korea.

ail.net (H.-S. Kim).

this context, transplantation of whole pancreas or primary isletsis ideal because it provides the physiologic glucose control withoutcomplications. But, shortage of matched islet-replacement tissueshas prevented the widespread use of this therapy [3]. Therefore,there is a need for alternative approaches for transplantation ofinsulin-secreting cells generated from ex-vivo expanded sources.Recent advances in stem cell biology have generated enthusiasmto apply stem cells to treatment of diverse ranges of humandiseases [4,5]. For treatment of T1DM, in the several studies, insulinproducing cells (IPCs) were differentiated and generated fromvarious kinds of stem cells including embryonic stem cells (ESCs)or mesenchymal stem cells (MSCs) [6–8]. However, IPCs generatedfrom those kinds of stem cells have serious hurdles to be appliedclinically. Actually, there has been not only ethical controversybut also immunological rejection on using ESCs. On the other hand,MSCs have some challenges such as limited obtainable amount andrestricted differentiation potency. Therefore, we need the easilyobtainable autologous cell sources. Peripheral blood mononuclearcells (PBMNCs) have several merits: they are autologous not to

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cause immunologic reaction and easy to obtain. We had previouslydeveloped the new protocol to make human blood born hemato-sphere (BBHS) through three-dimensional culture of PBMNCs anddemonstrated that BBHS consisted of myeloid cells and provideda stem cell niche for hematopoietic stem cells (HSCs) expansion[9]. Hence, in this study, we evaluated the potential of BBHS as anautologous source for human insulin producing cells by determin-ing whether insulin positive cells are located or induced in thisBBHS.

2. Materials and methods

2.1. Three-dimensional culture of hPBMC (BBHS culture)

All study protocols in this study were approved by the Institu-tional Review Board of Seoul National University Hospital. Freshhuman peripheral blood (hPB) was obtained from healthy donors.Human peripheral blood mononuclear cells (hPBMNCs) were iso-lated as described previously with slight modifications [10].Briefly, hPBMNCs were isolated by Ficoll-Paque™ PLUS (GE health-care) according to instructions by the manufacturer and werewashed five times with phosphate buffered saline (PBS) to com-pletely remove remaining platelets and debris hPBMNCs wereseeded and cultured at 2 � 107 cells in endothelial basal media-2(EBM-2) purchased from Lonza with 1% FBS on Hydrocell™ Ultra-Low attach surface (NUNC). One ml of EBM-2 with 1%FBS freshmedium was supplied every second day without media change.

2.2. Immunofluorescence analysis

Immunostaining was performed as described previously withminor modifications [9]. Briefly, differentiated BBHSs were fixedin 2% paraformaldehyde for 10 min and washed with PBS twice.To confirm insulin producing capacity by BBHS-derived IPCs, anti-bodies against insulin (Abcam, R&D Systems) were used. Nestin (aneural stem cell marker) and b-tubulin III (a neural cell marker)which were purchased from Millipore were used specific markers.The appropriate fluorescent-labeled secondary antibodies (1:1000)were from Invitrogen.

2.3. RNA extraction and RT-PCR analysis

Total RNA was extracted from fresh hPBMNCs or culturedBBHS-IPCs using TRIZOL reagent (Invitrogen) according to the man-ufacturer’s instruction. Reverse transcription PCR (RT-PCR) wasperformed as described previously [11]. In short, cDNA was synthe-sized using a Primescript 1st strand cDNA synthesis kit (TAKARA)and oligo-dT primer. Information of corresponding primers is pro-vided in Supplementary table 1.

2.4. IPCs differentiation protocol

We induced BBHS to differentiate into IPCs as described previ-ously with slight modification [12]. The IPCs protocol is dividedinto four distinct stages. Stage I: The 7 days-cultured undifferenti-ated BBHS in the suspension condition were cultivated in EBM-2supplemented with 1% FBS and low concentration (5.9 mM) of glu-cose (Sigma Aldrich) for two days in the attaching culture condi-tion on the dish (NUNC) coated with 5 lg/ml fibronectin (SigmaAldrich). Stage II: The media were replaced with same growthmedia plus high concentration (25 mM) of glucose and culturedfor two additional days. Stage III: The media was again replacedwith a growth media plus N2 supplement (Invitrogen) to stage Imedia and cultured for two additional days. Stage 4: The mediawas replaced with EBM-2, supplied with 1% FBS and 10 mM nico-

tinamide (Sigma Aldrich) in the present of N2 supplement, thencultured for two additional days.

2.5. Dithizone staining

The 7 days-BBHS that were induced to differentiate during allfour stages were attached on an ultra low attach dish. They werestained with zinc-chelating agent, Dithizone (DTZ) that was pur-chased from Sigma Aldrich and prepared by dissolving 50 mg ofDTZ in 5 ml of dimethyl sulfoxide (DMSO). In vitro DTZ stainingwas achieved by adding 10 ll of the stock solution (10 mg/ml) into1 ml of EBM-2 with 1% FBS growth media, according to distinct dif-ferentiation stage as described. The staining solution was filteredthrough a 0.2 lm nylon filter then used as the DTZ working solu-tion. The culture dishes were incubated at 37 �C for 15 min in thepresence of DTZ solution. After DTZ staining, the culture disheswere washed three times with Hank’s Balanced Salt Solutions. Fi-nally, cells or pellets stained with crimson red were examined witha stereomicroscope.

2.6. Glucose-stimulated insulin secretion assay (GSIS Assay)

The assay of insulin secretion level from glucose stimulatedBBHS-IPCs was performed as described previously with slightmodifications [13]. In brief, cells in stage IV were gently washedwith PBS once. Then, they were pre-incubated in Krebs-Ringerbicarbonate (KRB) buffer (120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2,1.1 mM MgCl2, 25 mM NaHCO3, 0.1 g BSA), containing low dose ofglucose (5.9 mM) in 37 �C for an hour. Supernatants from pre-incu-bated cells were harvested. New KRB buffer containing 5.9 or25 mM glucose was added into two separate groups of wells andincubated at 37 �C for 2 h. After 2 h incubation, supernatants frompost-incubated cells were harvested from wells containing 5.9 ver-sus 25 mM glucose. We measured insulin levels in pre-incubatedand post-incubated supernatants from cells exposed to low versushigh glucose condition using an enzyme-linked immunosorbantassay (ELISA), which detects human insulin (not pro-insulin) orc-peptide. The undifferentiated cells were used as control.

2.7. Statistical analysis

All results are expressed as means ± SD. p 6 0.05 was consid-ered significant.

3. Results

3.1. Generation of human hematospheres that have neuronalcharacteristics

As previously reported, we made progress to make BBHS withhigh density suspension culture system following the protocol.BBHS is a kind of optimal ex vivo niche to potentiate the expansionof HSCs [9]. The scheme of protocol was showed in SupplementaryFig. 1. After development of BBHS, we evaluated the potential ofBBHS as a source for insulin producing cells by screening theexistence of neural specific markers on BBHS through immunoflu-orescence, because one previous paper [12] demonstrated thathuman neural progenitor cells can differentiate into IPCs throughextracellular factor modification without manipulations of genes.We stained nestin (a neural stem cell marker) [14] and b-tubulinIII (a neural cell marker) [15] in the BBHS. Interestingly, nestin-positive and b-tubulin III-positive cells were found at the wholearea of hematospheres (Fig. 1A and B).

BA Nestin -tubulin III

InsulinC

Low Glucose(5.9mM) Fibronectin(5ug/mL)

High Glucose (25mM)Fibronectin(5ug/mL)

Low Glucose (5.9mM)Fibronectin(5ug/mL)N2 supplement

High Glucose(25mM)Fibronectin(5ug/mL)Nicotinamide + 1% EBM

2days 2days 2days 2days

Stage 1 Stage 2 Stage 3 Stage 47daysBBHS

Suspensionculture

Attaching culture

2daysD

Fig. 1. Generation of hematospheres with neuronal characteristics and IPCs differentiation using BBHS. (A and B) Nestin (a neural stem cell marker) and b-tubulin III (a neuralcell marker) positive cells are detected in the hematospheres via immunofluorescence. Scale bar represents 50 lm. (C) A few insulin-stained cells (green color) existed in theundifferentiated hematosphere. Scale bar represents 50 lm. (D) Schematic image represents the stepwise differentiation protocol to generate IPCs from BBHS. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.2. Insulin-positive cells in human BBHS and strategy to induce IPCs

To determine whether insulin-positive cells would exist inBBHS cultured on three-dimensional system, we performed confo-cal analysis (Fig. 1C). A few IPCs were observed at the external sideof BBHS (Fig. 1C). In our previous study [9], fibronectin and lamininpivotal to IPCs differentiation were abundant in BBHS generatedvia three-dimensional culture system. Thus we attempted toinduce the differentiation of IPCs out of BBHS, adopting thefour-stage protocol [12] following IPCs development protocol(Fig. 1D). Throughout the IPCs differentiation process, the numberof islet-like cluster increased resulting in the most plentifulislet-like clusters at stage 4 (Fig. 1D).

3.3. Induction of b cell differentiation using human BBHS

To determine IPCs development from BBHS, we performedimmunofluorescence analysis of islet-like clusters at stage 4. Insu-lin-positive cells constituted the most part of islet-like clusters andsome of them were also positive for nestin, suggesting that IPCswere differentiated from nestin-positive cells in BBHS duringfour-stage of differentiation (Fig. 2A).

To confirm whether cells in BBHS had undergone IPCs differen-tiation, expression of pancreatic b cell markers were assessed byRT-PCR at the four differentiation stages (Fig. 2B). Expression ofpancreatic transcription factor genes (Pdx-1, Ngn3 and Nkx6.1) in-creased throughout the differentiation process. Also genes related

Insulin

PDX-1

Glut-2

Ngn-3

Nkx6.1

PC2

PC1/3

IGF-1

Nestin

Sur-1

GAPDH

IPCs development stages

0 1 2 3 4

-1

-2

-3

-1

-1

-1

-2

-3

-1

-1

0 1 2 3 4

Nes

tin(g

reen

)In

sulin

(red

)

BBHS-derived IPCs

)

A

B

*

Insu

lin(m

IU/L

)

**

C

Fig. 2. BBHS-derived IPCs: pancreatic b cell specific gene expression and insulin secreted level. (A) We stained cells with insulin (red) and nestin (green) at stage 4 IPCsdifferentiation protocol through immunofluorescence. IPCs constitute the majority of the cell population of islet-like clusters derived from BBHS after completion of 4 stagesof differentiation for 8 days. Some of them are still positive for nestin, suggesting the possibility of differentiation of neural precursors to IPCs in BBHS. Scale bar represents50 lm. (B) Gene expression of pancreatic b cell markers was examined throughout the four-differentiation stages by RT-PCR analysis. Pancreatic transcription factor genes(Pdx-1, Ngn3 and Nkx6.1) increased throughout the differentiation process. Also increased genes related to pancreatic endocrine function (Glut-2 and PC2) or b cell function(Kir6.2, SUR1). (C) Secretion level of Insulin from IPCs-BBHS by GSIS assay was examined via ELISA by GSIS assay, demonstrating that IPCs from BBHS have capability tosecrete insulin in response to low (5.9 mM) and high (25.6 mM) glucose simulation. (Data represent mean ± SD, n = 3). (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

768 J. Hur et al. / Biochemical and Biophysical Research Communications 418 (2012) 765–769

to pancreatic endocrine function (Glut-2 and PC2) or b cell function(Kir6.2, SUR1) showed increasing expression level (Fig. 2B). How-ever, IGF-1 and nestin gene expression had reduced during IPCsdevelopment as did PC1/3 (Fig. 2B).

3.4. Assessment of insulin secretion in IPCs originating from humanBBHS

IPCs differentiation was further evaluated through DTZ stain-ing to detect the binding of zinc ion to insulin molecules withinIPCs. We confirmed that insulin expression increased throughoutthe distinct four-stage processes (Supplementary Fig. 2). Next, weemployed GSIS assay to further examine the capability of IPCs

from human BBHS to secrete insulin in response to glucose stim-ulation. As Fig. 2C depicted, IPCs from BBHS showed up-regulatedresponse to high glucose stimulation (25 mM) by secreting insulinat concentration of 4.2 ± 0.62 (mIU/L) that is about 3.5-fold higherthan insulin level by IPCs in response to low glucose (5.9 mM)stimulation. In summary, secretion of insulin from IPCs-BBHSwas stimulated by high glucose, representing the ‘glucoseresponsiveness’.

4. Discussion

In this study, we have developed the strategy to generate thehuman insulin producing cells through three-dimensional culture

J. Hur et al. / Biochemical and Biophysical Research Communications 418 (2012) 765–769 769

system using human peripheral blood cells. Recently, it wasreported that human nestin-positive neural progenitor cellscould be differentiated into IPCs through modification of extra-cellular factors [12]. Interestingly, our BBHS contained numerousnestin-positive cells and abundant extracellular matrix such asfibronectin and laminin as important factors for the differentia-tion of IPCs from neural progenitor cells. Through correlationbetween previous study and ours, we could set up a newhypothesis that nestin-positive cells in BBHS may undergodifferentiation into IPCs. To our expectation, under the stageddifferentiation protocol, we were able to get a lot of human IPCsfrom BBHS that showed capability to secrete insulin in responseto glucose stimulation.

Which factors are important to make optimal niche for IPCsdifferentiation in BBHS? Suspension culture of pellets of MSCson fibronectin enhanced IPCs differentiation [16]. Similarly, ourBBHS culture system provides a suspension environment andabundant fibronectin [9]. Through this, we could infer that the3D-suspension state with fibronectin is important for differentia-tion of IPCs from BBHS. Further study should be investigated indetail.

Is the insulin secreted from IPCs of BBHS functional? We have tolook at our findings from a new angle based on the research thathuman blood myeloid cells express pro-insulin on their surfaceas a self-antigen [17]. From the perspective that human blood mye-loid cells may be associated with peripheral immune tolerance[17], it is possible that insulin detected in IPCs derived from BBHSmay be just self-antigen of blood myeloid cells. Therefore, in thefuture we have to elucidate whether insulin detected in IPCs ofBBHS is just surface antigen or is actually produced to control glu-cose level.

Although our IPCs from BBHS showed the glucose-responsiveinsulin secretion level was low, implying that specific measuresshould be developed to improve the efficiency of insulin secretionbefore clinical application. Another limitation in our study is thatwe actually did not detect C-peptide by ELISA of the culture med-ia during each IPCs differentiation stage (data not shown). Thereare some possibilities that secretary mechanisms or insulin pro-cessing machines of IPCs would be in a dysfunctional state. How-ever, previous research [12] also showed that C-peptide is usuallyundetectable in vitro assay, but it would be possible to confirm C-peptide existence in vivo assay. Thus, more investigations shouldbe performed not only for increasing the level of insulin expres-sion but also for understanding insulin secretion mechanisms inIPCs from BBHS. Furthermore, study for feasibility to regulatethe blood glucose level should be investigated in vivo diabetesmellitus model.

Regenerative medicine using stem cell biology provides newperspectives of future clinical practice for us [18] and chancesto overcome several incurable diseases [19]. Up to date, thereare several stem cell sources to be applied for regenerative med-icine such as ESCs, the induced pluripotent stem cells (iPCs), andadult stem cells like MSCs or HSCs etc. Compared to them, ourBBHS has the several advantages distinguishing itself from theothers: ‘‘easily-obtainable autologous cell source’’. If we coulddiscover a new mechanism to boost insulin expression andsecretion in IPCs-BBHS, it would be a very useful treatmentsource for DM.

Taken together, our findings clearly demonstrate the potentialof human BBHS with abundant nestin-positive cells to differentiateinto insulin producing cells. Our novel strategy of ex vivo expan-sion of IPCs using BBHS provides an autologous therapeutic cellsource for the treatment of diabetes.

Acknowledgments

This study was supported by grants from the National ResearchFoundation funded by the Ministry of Education, Science, andTechnology (MEST), Republic of Korea 2010-0020257. Dr. Hyo-Soo Kim is also a professor of Molecular Medicine and Biopharma-ceutical Sciences, Seoul National University, sponsored by theWorld Class University program of MEST, Republic of Korea.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2012.01.096.

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