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

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

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New method to differentiate human peripheral blood monocytes into insulin producing 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 Korea Innovative Research Institute for Cell Therapy, Seoul National University, 101 Daehak-ro, JongRo-gu, Seoul, Republic of Korea Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea d Cardiovascular Center, Seoul National University Bundang Hospital, 173 Beon gil, Bundang gu, Seongnam, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 18 January 2012 Available online 28 January 2012 Keywords: Three-dimensional culture Insulin Insulin producing cells Stem cells Spheroid Regenerative medicine

a b s t r a c t Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of b-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a threedimensional 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-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form 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-2 and 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 from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Diabetes mellitus (DM) has been widespread all around the world affecting more than one hundred million patients, and it is expected to increase dramatically in the next decade [1,2]. Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease in which insulin-secreting b-cells are irreversibly destroyed, resulting in deficiency of circulating insulin [3]. To date administration of exogenous insulin remains the main treatment in T1DM [1]. However, the exogenous insulin injection is not physiologic and inevitably cause serious complications such as hypoglycemia. In Abbreviations: FBS, fetal bovine serum; DTZ, dithizone; EBM-2, endothelial basal media 2; DMSO, dimethyl sulfoxide; hPB, human peripheral blood; hPBMNCs, human peripheral blood mononuclear cells; Glc, glucose; BBHS, blood-born hematosphere; NPCs, nestin-positive cells; IPCs, insulin-producing cells. ⇑ Corresponding author at: Department of Internal Medicine, Seoul National University Hospital, 101 DaeHak-ro, JongRo-gu, Seoul 110-744, Republic of Korea. Fax: +82 2 766 8904. E-mail addresses: [email protected], [email protected] (H.-S. Kim). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.096

this context, transplantation of whole pancreas or primary islets is ideal because it provides the physiologic glucose control without complications. But, shortage of matched islet-replacement tissues has prevented the widespread use of this therapy [3]. Therefore, there is a need for alternative approaches for transplantation of insulin-secreting cells generated from ex-vivo expanded sources. Recent advances in stem cell biology have generated enthusiasm to apply stem cells to treatment of diverse ranges of human diseases [4,5]. For treatment of T1DM, in the several studies, insulin producing cells (IPCs) were differentiated and generated from various kinds of stem cells including embryonic stem cells (ESCs) or mesenchymal stem cells (MSCs) [6–8]. However, IPCs generated from those kinds of stem cells have serious hurdles to be applied clinically. Actually, there has been not only ethical controversy but also immunological rejection on using ESCs. On the other hand, MSCs have some challenges such as limited obtainable amount and restricted differentiation potency. Therefore, we need the easily obtainable autologous cell sources. Peripheral blood mononuclear cells (PBMNCs) have several merits: they are autologous not to

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cause immunologic reaction and easy to obtain. We had previously developed the new protocol to make human blood born hematosphere (BBHS) through three-dimensional culture of PBMNCs and demonstrated that BBHS consisted of myeloid cells and provided a stem cell niche for hematopoietic stem cells (HSCs) expansion [9]. Hence, in this study, we evaluated the potential of BBHS as an autologous source for human insulin producing cells by determining whether insulin positive cells are located or induced in this BBHS. 2. Materials and methods 2.1. Three-dimensional culture of hPBMC (BBHS culture) All study protocols in this study were approved by the Institutional Review Board of Seoul National University Hospital. Fresh human peripheral blood (hPB) was obtained from healthy donors. Human peripheral blood mononuclear cells (hPBMNCs) were isolated as described previously with slight modifications [10]. Briefly, hPBMNCs were isolated by Ficoll-Paque™ PLUS (GE healthcare) according to instructions by the manufacturer and were washed five times with phosphate buffered saline (PBS) to completely remove remaining platelets and debris hPBMNCs were seeded and cultured at 2  107 cells in endothelial basal media-2 (EBM-2) purchased from Lonza with 1% FBS on Hydrocell™ UltraLow attach surface (NUNC). One ml of EBM-2 with 1%FBS fresh medium was supplied every second day without media change.

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

2.5. Dithizone staining The 7 days-BBHS that were induced to differentiate during all four stages were attached on an ultra low attach dish. They were stained with zinc-chelating agent, Dithizone (DTZ) that was purchased from Sigma Aldrich and prepared by dissolving 50 mg of DTZ in 5 ml of dimethyl sulfoxide (DMSO). In vitro DTZ staining was achieved by adding 10 ll of the stock solution (10 mg/ml) into 1 ml of EBM-2 with 1% FBS growth media, according to distinct differentiation stage as described. The staining solution was filtered through a 0.2 lm nylon filter then used as the DTZ working solution. The culture dishes were incubated at 37 °C for 15 min in the presence of DTZ solution. After DTZ staining, the culture dishes were washed three times with Hank’s Balanced Salt Solutions. Finally, cells or pellets stained with crimson red were examined with a stereomicroscope.

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

Immunostaining was performed as described previously with minor modifications [9]. Briefly, differentiated BBHSs were fixed in 2% paraformaldehyde for 10 min and washed with PBS twice. To confirm insulin producing capacity by BBHS-derived IPCs, antibodies against insulin (Abcam, R&D Systems) were used. Nestin (a neural 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.

The assay of insulin secretion level from glucose stimulated BBHS-IPCs was performed as described previously with slight modifications [13]. In brief, cells in stage IV were gently washed with PBS once. Then, they were pre-incubated in Krebs-Ringer bicarbonate (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 of glucose (5.9 mM) in 37 °C for an hour. Supernatants from pre-incubated cells were harvested. New KRB buffer containing 5.9 or 25 mM glucose was added into two separate groups of wells and incubated at 37 °C for 2 h. After 2 h incubation, supernatants from post-incubated cells were harvested from wells containing 5.9 versus 25 mM glucose. We measured insulin levels in pre-incubated and post-incubated supernatants from cells exposed to low versus high glucose condition using an enzyme-linked immunosorbant assay (ELISA), which detects human insulin (not pro-insulin) or c-peptide. The undifferentiated cells were used as control.

2.3. RNA extraction and RT-PCR analysis

2.7. Statistical analysis

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

All results are expressed as means ± SD. p 6 0.05 was considered significant.

2.2. Immunofluorescence analysis

3. Results 3.1. Generation of human hematospheres that have neuronal characteristics

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

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

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

Nestin

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˟-tubulin III

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Insulin C

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Suspension culture 7days BBHS

Attaching culture 2days

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Stage 2

Stage 3

Stage 4

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

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 neural cell 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 the undifferentiated hematosphere. Scale bar represents 50 lm. (D) Schematic image represents the stepwise differentiation protocol to generate IPCs from BBHS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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

3.3. Induction of b cell differentiation using human BBHS

To determine whether insulin-positive cells would exist in BBHS cultured on three-dimensional system, we performed confocal analysis (Fig. 1C). A few IPCs were observed at the external side of BBHS (Fig. 1C). In our previous study [9], fibronectin and laminin pivotal to IPCs differentiation were abundant in BBHS generated via three-dimensional culture system. Thus we attempted to induce the differentiation of IPCs out of BBHS, adopting the four-stage protocol [12] following IPCs development protocol (Fig. 1D). Throughout the IPCs differentiation process, the number of islet-like cluster increased resulting in the most plentiful islet-like clusters at stage 4 (Fig. 1D).

To determine IPCs development from BBHS, we performed immunofluorescence analysis of islet-like clusters at stage 4. Insulin-positive cells constituted the most part of islet-like clusters and some of them were also positive for nestin, suggesting that IPCs were differentiated from nestin-positive cells in BBHS during four-stage of differentiation (Fig. 2A). To confirm whether cells in BBHS had undergone IPCs differentiation, expression of pancreatic b cell markers were assessed by RT-PCR at the four differentiation stages (Fig. 2B). Expression of pancreatic transcription factor genes (Pdx-1, Ngn3 and Nkx6.1) increased throughout the differentiation process. Also genes related

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

BBHS-derived IPCs

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Insulin(red))

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4 Insulin Glut-- 2

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Ngn-- 3 Nkx6.1 PC2

Insulin(mIU/L)

PDX -1

PC1/3 -1 Sur-1 -1 IGF-1 Nestin GAPDH 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 IPCs differentiation protocol through immunofluorescence. IPCs constitute the majority of the cell population of islet-like clusters derived from BBHS after completion of 4 stages of 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 represents 50 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 to secrete 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 this figure legend, the reader is referred to the web version of this article.)

to pancreatic endocrine function (Glut-2 and PC2) or b cell function (Kir6.2, SUR1) showed increasing expression level (Fig. 2B). However, IGF-1 and nestin gene expression had reduced during IPCs development as did PC1/3 (Fig. 2B). 3.4. Assessment of insulin secretion in IPCs originating from human BBHS IPCs differentiation was further evaluated through DTZ staining to detect the binding of zinc ion to insulin molecules within IPCs. We confirmed that insulin expression increased throughout the distinct four-stage processes (Supplementary Fig. 2). Next, we employed GSIS assay to further examine the capability of IPCs

from human BBHS to secrete insulin in response to glucose stimulation. As Fig. 2C depicted, IPCs from BBHS showed up-regulated response to high glucose stimulation (25 mM) by secreting insulin at concentration of 4.2 ± 0.62 (mIU/L) that is about 3.5-fold higher than insulin level by IPCs in response to low glucose (5.9 mM) stimulation. In summary, secretion of insulin from IPCs-BBHS was stimulated by high glucose, representing the ‘glucose responsiveness’. 4. Discussion In this study, we have developed the strategy to generate the human insulin producing cells through three-dimensional culture

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

system using human peripheral blood cells. Recently, it was reported that human nestin-positive neural progenitor cells could be differentiated into IPCs through modification of extracellular factors [12]. Interestingly, our BBHS contained numerous nestin-positive cells and abundant extracellular matrix such as fibronectin and laminin as important factors for the differentiation of IPCs from neural progenitor cells. Through correlation between previous study and ours, we could set up a new hypothesis that nestin-positive cells in BBHS may undergo differentiation into IPCs. To our expectation, under the staged differentiation protocol, we were able to get a lot of human IPCs from BBHS that showed capability to secrete insulin in response to glucose stimulation. Which factors are important to make optimal niche for IPCs differentiation in BBHS? Suspension culture of pellets of MSCs on fibronectin enhanced IPCs differentiation [16]. Similarly, our BBHS culture system provides a suspension environment and abundant fibronectin [9]. Through this, we could infer that the 3D-suspension state with fibronectin is important for differentiation of IPCs from BBHS. Further study should be investigated in detail. Is the insulin secreted from IPCs of BBHS functional? We have to look at our findings from a new angle based on the research that human blood myeloid cells express pro-insulin on their surface as a self-antigen [17]. From the perspective that human blood myeloid cells may be associated with peripheral immune tolerance [17], it is possible that insulin detected in IPCs derived from BBHS may be just self-antigen of blood myeloid cells. Therefore, in the future we have to elucidate whether insulin detected in IPCs of BBHS is just surface antigen or is actually produced to control glucose level. Although our IPCs from BBHS showed the glucose-responsive insulin secretion level was low, implying that specific measures should be developed to improve the efficiency of insulin secretion before clinical application. Another limitation in our study is that we actually did not detect C-peptide by ELISA of the culture media during each IPCs differentiation stage (data not shown). There are some possibilities that secretary mechanisms or insulin processing machines of IPCs would be in a dysfunctional state. However, previous research [12] also showed that C-peptide is usually undetectable in vitro assay, but it would be possible to confirm Cpeptide existence in vivo assay. Thus, more investigations should be performed not only for increasing the level of insulin expression but also for understanding insulin secretion mechanisms in IPCs from BBHS. Furthermore, study for feasibility to regulate the blood glucose level should be investigated in vivo diabetes mellitus model. Regenerative medicine using stem cell biology provides new perspectives of future clinical practice for us [18] and chances to overcome several incurable diseases [19]. Up to date, there are several stem cell sources to be applied for regenerative medicine such as ESCs, the induced pluripotent stem cells (iPCs), and adult stem cells like MSCs or HSCs etc. Compared to them, our BBHS has the several advantages distinguishing itself from the others: ‘‘easily-obtainable autologous cell source’’. If we could discover a new mechanism to boost insulin expression and secretion in IPCs-BBHS, it would be a very useful treatment source for DM. Taken together, our findings clearly demonstrate the potential of human BBHS with abundant nestin-positive cells to differentiate into insulin producing cells. Our novel strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes.

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Acknowledgments This study was supported by grants from the National Research Foundation funded by the Ministry of Education, Science, and Technology (MEST), Republic of Korea 2010-0020257. Dr. HyoSoo Kim is also a professor of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, sponsored by the World Class University program of MEST, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.01.096. References [1] D. Devendra, E. Liu, G.S. Eisenbarth, Type 1 diabetes: recent developments, BMJ 328 (2004) 750–754. [2] H.W. Park, T.G. Kown, K.Y. Kim, J.H. Bae, Diabetes, insulin resistance and atherosclerosis surrogates in patients with coronary atherosclerosis, Korean Circ. J. 40 (2010) 62–67. [3] P.M. Jones, M.L. Courtney, C.J. Burns, S.J. Persaud, Cell-based treatments for diabetes, Drug. Discov. Today. 13 (2008) 888–893. [4] C.J. Taylor, E.M. Bolton, J.A. Bradley, Immunological considerations for embryonic and induced pluripotent stem cell banking, Philos. Trans. R Soc. Lond. B Biol. Sci. 366 (2011) 2312–2322. [5] K.H. Byun, S.W. Kim, Is stem cell-based therapy going on or out for cardiac disease? Korean Circ. J. 39 (2009) 87–92. [6] A.S. Boyd, K.J. Wood, Characteristics of the early immune response following transplantation of mouse ES cell derived insulin-producing cell clusters, PLoS ONE 5 (2010) e10965. [7] B.E. Tuch, T.C. Hughes, M.D. Evans, Encapsulated pancreatic progenitors derived from human embryonic stem cells as a therapy for insulindependent diabetes, Diabetes Metab. Res. Rev. 27 (2011) 928–932. [8] H. Motoyama, S. Ogawa, A. Kubo, S. Miwa, J. Nakayama, Y. Tagawa, S. Miyagawa, In vitro reprogramming of adult hepatocytes into insulinproducing cells without viral vectors, Biochem. Biophys. Res. Commun. 385 (2009) 123–128. [9] J. Hur, J. Park, S.E. Lee, C.H. Yoon, J.H. Jang, J.M. Yang, T.K. Lee, J.I. Choi, H.M. Yang, E.J. Lee, H.J. Cho, H.J. Kang, B.H. Oh, Y.B. Park, H.S. Kim, Human peripheral blood-born hematosphere as a niche for hematopoietic stem cell expansion, Cell Res. 21 (2011) 987–990. [10] J. Hur, H.M. Yang, C.H. Yoon, C.S. Lee, K.W. Park, J.H. Kim, T.Y. Kim, J.Y. Kim, H.J. Kang, I.H. Chae, B.H. Oh, Y.B. Park, H.S. Kim, Identification of a novel role of T cells in postnatal vasculogenesis – Characterization of endothelial progenitor cell colonies, Circulation 116 (2007) 1671–1682. [11] J. Hur, C.H. Yoon, H.S. Kim, J.H. Choi, H.J. Kang, K.K. Hwang, B.H. Oh, M.M. Lee, Y.B. Park, Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 288–293. [12] Y. Hori, X. Gu, X. Xie, S.K. Kim, Differentiation of insulin-producing cells from human neural progenitor cells, PLoS Med. 2 (2005) e103. [13] H.E. Hohmeier, H. Mulder, G. Chen, R. Henkel-Rieger, M. Prentki, C.B. Newgard, Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channeldependent and -independent glucose-stimulated insulin secretion, Diabetes 49 (2000) 424–430. [14] J.F. Bond, G.S. Robinson, S.R. Farmer, Differential expression of two neural cellspecific beta-tubulin mRNAs during rat brain development, Mol. Cell. Biol. 4 (1984) 1313–1319. [15] J. Drapeau, V. El-Helou, R. Clement, S. Bel-Hadj, H. Gosselin, L.E. Trudeau, L. Villeneuve, A. Calderone, Nestin-expressing neural stem cells identified in the scar following myocardial infarction, J. Cell. Physiol. 204 (2005) 51–62. [16] C.F. Chang, K.H. Hsu, S.H. Chiou, L.L. Ho, Y.S. Fu, S.C. Hung, Fibronectin and pellet suspension culture promote differentiation of human mesenchymal stem cells into insulin producing cells, J. Biomed. Mater Res. A 86 (2008) 1097–1105. [17] P. Narendran, A.M. Neale, B.H. Lee, K. Ngui, R.J. Steptoe, G. Morahan, O. Madsen, J.A. Dromey, K.P. Jensen, L.C. Harrison, Proinsulin is encoded by an RNA splice variant in human blood myeloid cells, Proc. Natl. Acad Sci. USA 103 (2006) 16430–16435. [18] G.B. Tomar, R.K. Srivastava, N. Gupta, A.P. Barhanpurkar, S.T. Pote, H.M. Jhaveri, G.C. Mishra, M.R. Wani, Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine, Biochem. Biophys. Res. Commun. 393 (2010) 377–383. [19] T.J. Nelson, A. Martinez-Fernandez, S. Yamada, Y. Ikeda, C. Perez-Terzic, A. Terzic, Induced pluripotent stem cells: advances to applications, Stem Cells Cloning 3 (2010) 29–37.