Continuous Inhibition of Sonic Hedgehog Signaling Leads to Differentiation of Human-Induced Pluripotent Stem Cells into Functional Insulin-Producing β Cells
Song Lee
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorJae Hyun Joo
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorJu Yun Oh
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorEun Ha Seo
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorYang Hee Kim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorEunsung Jun
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorIn Kyong Shim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorCorresponding Author
Song Cheol Kim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorSong Lee
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorJae Hyun Joo
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorJu Yun Oh
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorEun Ha Seo
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorYang Hee Kim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorEunsung Jun
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorIn Kyong Shim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorCorresponding Author
Song Cheol Kim
Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Department of Medicine, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea ulsan.ac.kr
Search for more papers by this authorAbstract
Human-induced pluripotent stem cell- (iPSC-) derived insulin-producing cells (IPCs) can be used for islet cell transplantation into type 1 diabetic patients and as patient-specific cells for the development of novel antidiabetic drugs. However, a method is needed to generate functional IPCs from iPSCs and simplify the protocol. We compared combinations of small molecules that could induce the differentiation of cells into a definitive endoderm and preferentially into islet precursor cells. When generated using an optimal combination of small molecules, IPCs secreted insulin in response to glucose stimulation. We constructed spheroid IPCs and optimized the culture and maturation conditions. Quantitative PCR revealed that the expression of definitive endoderm-specific markers differed depending on the combination of the small molecules. The small molecule, N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine, induced the differentiation of cells into functional IPCs by inhibiting Sonic hedgehog signaling. Images of the 2D culture showed that IPCs formed spheroids from day 5 and continuously secreted insulin. We developed a simple differentiation method using small molecules that produced functional IPCs that responded to glucose stimulation within a relatively short period. We posit that this method along with further refinement of the differentiation process can be applied to culture IPCs that can be used in clinical trials.
Conflicts of Interest
The authors declare that they have no conflicting interests.
Open Research
Data Availability
The data used to support the findings of this study are included within the article.
References
- 1 Schiesser J. V., Micallef S. J., Hawes S., Elefanty A. G., and Stanley E. G., Derivation of insulin-producing Beta-cells from human pluripotent stem cells, The Review of Diabetic Studies. (2014) 11, no. 1, 6–18, https://doi.org/10.1900/RDS.2014.11.6, 2-s2.0-84906345800, 25148364.
- 2 Yabe S. G., Fukuda S., Takeda F., Nashiro K., Shimoda M., and Okochi H., Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells, Journal of Diabetes. (2017) 9, no. 2, 168–179, https://doi.org/10.1111/1753-0407.12400, 2-s2.0-85008466229, 27038181.
- 3 Jiang J., Au M., Lu K., Eshpeter A., Korbutt G., Fisk G., and Majumdar A. S., Generation of insulin-producing islet-like clusters from human embryonic stem cells, Stem Cells. (2007) 25, no. 8, 1940–1953, https://doi.org/10.1634/stemcells.2006-0761, 2-s2.0-34547857040, 17510217.
- 4 Zhang D., Jiang W., Liu M., Sui X., Yin X., Chen S., Shi Y., and Deng H., Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells, Cell Research. (2009) 19, no. 4, 429–438, https://doi.org/10.1038/cr.2009.28, 2-s2.0-64149118087, 19255591.
- 5 Jiang W., Shi Y., Zhao D., Chen S., Yong J., Zhang J., Qing T., Sun X., Zhang P., Ding M., Li D., and Deng H., In vitro derivation of functional insulin-producing cells from human embryonic stem cells, Cell Research. (2007) 17, no. 4, 333–344, https://doi.org/10.1038/cr.2007.28, 2-s2.0-34247353657, 17426693.
- 6 D′Amour K. A., Bang A. G., Eliazer S., Kelly O. G., Agulnick A. D., Smart N. G., Moorman M. A., Kroon E., Carpenter M. K., and Baetge E. E., Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells, Nature Biotechnology. (2006) 24, no. 11, 1392–1401, https://doi.org/10.1038/nbt1259, 2-s2.0-33750846133, 17053790.
- 7 Kroon E., Martinson L. A., Kadoya K., Bang A. G., Kelly O. G., Eliazer S., Young H., Richardson M., Smart N. G., Cunningham J., Agulnick A. D., D′Amour K. A., Carpenter M. K., and Baetge E. E., Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo, Nature Biotechnology. (2008) 26, no. 4, 443–452, https://doi.org/10.1038/nbt1393, 2-s2.0-41849151748.
- 8 Segev H., Fishman B., Ziskind A., Shulman M., and Itskovitz-Eldor J., Differentiation of human embryonic stem cells into insulin-producing clusters, Stem Cells. (2004) 22, no. 3, 265–274, https://doi.org/10.1634/stemcells.22-3-265, 2-s2.0-2942615532, 15153604.
- 9 Kunisada Y., Tsubooka-Yamazoe N., Shoji M., and Hosoya M., Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells, Stem Cell Research. (2012) 8, no. 2, 274–284, https://doi.org/10.1016/j.scr.2011.10.002, 2-s2.0-84856085947, 22056147.
- 10 Chen S., Borowiak M., Fox J. L., Maehr R., Osafune K., Davidow L., Lam K., Peng L. F., Schreiber S. L., Rubin L. L., and Melton D., A small molecule that directs differentiation of human ESCs into the pancreatic lineage, Nature Chemical Biology. (2009) 5, no. 4, 258–265, https://doi.org/10.1038/nchembio.154, 2-s2.0-62649138086, 19287398.
- 11 Borowiak M., Maehr R., Chen S., Chen A. E., Tang W., Fox J. L., Schreiber S. L., and Melton D. A., Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells, Cell Stem Cell. (2009) 4, no. 4, 348–358, https://doi.org/10.1016/j.stem.2009.01.014, 2-s2.0-62949210744, 19341624.
- 12 Walczak M. P., Drozd A. M., Stoczynska-Fidelus E., Rieske P., and Grzela D. P., Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors, Journal of Translational Medicine. (2016) 14, no. 1, https://doi.org/10.1186/s12967-016-1097-0, 2-s2.0-85006746748, 27998294.
- 13 Fares I., Rivest-Khan L., Cohen S., and Sauvageau G., Small molecule regulation of normal and leukemic stem cells, Current Opinion in Hematology. (2015) 22, no. 4, 309–316, https://doi.org/10.1097/MOH.0000000000000151, 2-s2.0-84942811534, 26049751.
- 14 Zhang Y., Li W., Laurent T., and Ding S., Small molecules, big roles - the chemical manipulation of stem cell fate and somatic cell reprogramming, Journal of Cell Science. (2012) 125, no. 23, 5609–5620, https://doi.org/10.1242/jcs.096032, 2-s2.0-84874669151.
- 15 Shan J., Schwartz R. E., Ross N. T., Logan D. J., Thomas D., Duncan S. A., North T. E., Goessling W., Carpenter A. E., and Bhatia S. N., Identification of small molecules for human hepatocyte expansion and iPS differentiation, Nature Chemical Biology. (2013) 9, no. 8, 514–520, https://doi.org/10.1038/nchembio.1270, 2-s2.0-84880916761, 23728495.
- 16 Tasnim F., Phan D., Toh Y. C., and Yu H., Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules, Biomaterials. (2015) 70, 115–125, https://doi.org/10.1016/j.biomaterials.2015.08.002, 2-s2.0-84940869392, 26310107.
- 17 Osakada F., Jin Z. B., Hirami Y., Danjyo T., Watanabe K., Sasai Y., Takahashi M., and Takahashi M., In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction, Journal of Cell Science. (2009) 122, no. 17, 3169–3179, https://doi.org/10.1242/jcs.050393, 2-s2.0-70349347145.
- 18 Liu J., Liu Y., Wang H., Hao H., Han Q., Shen J., Shi J., Li C., Mu Y., and Han W., Direct differentiation of hepatic stem-like WB cells into insulin-producing cells using small molecules, Scientific Reports. (2013) 3, no. 1, https://doi.org/10.1038/srep01185, 2-s2.0-84873656615.
- 19 Huangfu D., Osafune K., Maehr R., Guo W., Eijkelenboom A., Chen S., Muhlestein W., and Melton D. A., Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2, Nature Biotechnology. (2008) 26, no. 11, 1269–1275, https://doi.org/10.1038/nbt.1502, 2-s2.0-55749104227, 18849973.
- 20 Takeuchi H., Nakatsuji N., and Suemori H., Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3Dculture, Scientific Reports. (2014) 27, no. 4.
- 21 Dhawan S., Dirice E., Kulkarni R. N., and Bhushan A., Inhibition of TGF-β signaling promotes human pancreatic β-cell replication, Diabetes. (2016) 65, no. 5, 1208–1218, https://doi.org/10.2337/db15-1331, 2-s2.0-84964573479, 26936960.
- 22 du J., Wu Y., Ai Z., Shi X., Chen L., and Guo Z., Mechanism of SB431542 in inhibiting mouse embryonic stem cell differentiation, Cellular Signalling. (2014) 26, no. 10, 2107–2116, https://doi.org/10.1016/j.cellsig.2014.06.002, 2-s2.0-84904416128, 24949833.
- 23 Nostro M. C., Sarangi F., Yang C., Holland A., Elefanty A. G., Stanley E. G., Greiner D. L., and Keller G., Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines, Stem Cell Reports. (2015) 4, no. 4, 591–604, https://doi.org/10.1016/j.stemcr.2015.02.017, 2-s2.0-84933677431, 25843049.
- 24 Pagliuca F. W., Millman J. R., Gürtler M., Segel M., van Dervort A., Ryu J. H., Peterson Q. P., Greiner D., and Melton D. A., Generation of functional human pancreatic β cells in vitro, Cell. (2014) 159, no. 2, 428–439, https://doi.org/10.1016/j.cell.2014.09.040, 2-s2.0-84910673362, 25303535.
- 25 Rezania A., Bruin J. E., Arora P., Rubin A., Batushansky I., Asadi A., O′Dwyer S., Quiskamp N., Mojibian M., Albrecht T., Yang Y. H. C., Johnson J. D., and Kieffer T. J., Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells, Nature Biotechnology. (2014) 32, no. 11, 1121–1133, https://doi.org/10.1038/nbt.3033, 2-s2.0-84983134468, 25211370.
- 26 Rostovskaya M., Bredenkamp N., and Smith A., Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells, Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. (2015) 370, no. 1680, https://doi.org/10.1098/rstb.2014.0365, 2-s2.0-84942920867, 26416676.
- 27 Ohori M., Kinoshita T., Okubo M., Sato K., Yamazaki A., Arakawa H., Nishimura S., Inamura N., Nakajima H., Neya M., Miyake H., and Fujii T., Identification of a selective ERK inhibitor and structural determination of the inhibitor-ERK2 complex, Biochemical and Biophysical Research Communications. (2005) 336, no. 1, 357–363, https://doi.org/10.1016/j.bbrc.2005.08.082, 2-s2.0-24344453386, 16139248.
- 28 Öström M., Loffler K. A., Edfalk S., Selander L., Dahl U., Ricordi C., Jeon J., Correa-Medina M., Diez J., and Edlund H., Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells, PLoS One. (2008) 3, no. 7, article e2841, https://doi.org/10.1371/journal.pone.0002841, 2-s2.0-50949102657, 18665267.
- 29 Cai J., Yu C., Liu Y., Chen S., Guo Y., Yong J., Lu W., Ding M., and Deng H., Generation of homogeneous PDX1(+) pancreatic progenitors from human ES cell-derived endoderm cells, Journal of Molecular Cell Biology. (2010) 2, no. 1, 50–60, https://doi.org/10.1093/jmcb/mjp037, 2-s2.0-77950364568, 19910415.
- 30 Johannesson M., Ståhlberg A., Ameri J., Sand F. W., Norrman K., and Semb H., FGF4 and retinoic acid direct differentiation of hESCs into PDX1-expressing foregut endoderm in a time- and concentration-dependent manner, PLoS One. (2009) 4, no. 3, article e4794, https://doi.org/10.1371/journal.pone.0004794, 2-s2.0-62849098510, 19277121.
- 31 Abazari M. F., Soleimanifar F., Nouri Aleagha M., Torabinejad S., Nasiri N., Khamisipour G., Amini Mahabadi J., Mahboudi H., Enderami S. E., Saburi E., Hashemi J., and Kehtari M., PCL/PVA nanofibrous scaffold improve insulin-producing cells generation from human induced pluripotent stem cells, Gene. (2018) 671, no. 671, 50–57, https://doi.org/10.1016/j.gene.2018.05.115, 2-s2.0-85047854832, 29860065.
- 32 Mansour R. N., Soleimanifar F., Abazari M. F., Torabinejad S., Ardeshirylajimi A., Ghoraeian P., Mousavi S. A., Sharif Rahmani E., Hassannia H., and Enderami S. E., Collagen coated electrospun polyethersulfon nanofibers improved insulin producing cells differentiation potential of human induced pluripotent stem cells, Artificial Cells, Nanomedicine, and Biotechnology. (2018) 46, no. sup3 Supplement 3, S734–S739, https://doi.org/10.1080/21691401.2018.1508031, 2-s2.0-85054503193.
- 33 Enderami S. E., Mortazavi Y., Soleimani M., Nadri S., Biglari A., and Mansour R. N., Generation of insulin-producing cells from human-induced pluripotent stem cells using a stepwise differentiation protocol optimized with platelet-rich plasma, Journal of Cellular Physiology. (2017) 232, no. 10, 2878–2886, https://doi.org/10.1002/jcp.25721, 2-s2.0-85018650476, 27925205.
