stem cell dan perannya di masa depan
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PENDAHULUAN Pada dekade terakhir perhatian dan penelitian dalam bidang sel punca (stem cells) mengalami kemajuan yang amat pesat. Para peneliti menggunakan sel punca untuk mengetahui dan mempelajari proses pertumbuhan dan perkembangan jaringan tubuh manusia serta patogenesis penyakit-penyakit yang diderita. Disamping itu penggunaan sel punca dalam perngobatan penyakit-penyakit yang sudah tidak mungkin untuk diobati lagi baik secara konservatif maupun operatif khususnya penyakit degeneratif maupun kelainan lainnya seperti trauma, keganasan dan sebagainya juga meningkat pesat. Dalam bidang farmakologi para peneliti juga menggunakan sel punca untuk menguji obat-obat baru. Tentu saja penggunaan sel punca dalam bidang penelitian dan pengobatan penyakit ini tidak terlepas dari potensi nilai bisnis yang akan diraih manakala sel punca ini sudah dapat digunakan untuk mengobati penyakit-penyakit atau kelainan-kelainan pada manusia. Penggunaan dan pengembangan sel punca dalam bidang penelitian dan aplikasinya diklinik dalam rangka mengobati penyakit tidak terlepas dari masalah etik yang mungkin membayanginya, khususnya penggunaan dan pemanfaatan sel punca yang berasal dari embrio (embryonic stem cells). Tanggal 12 Pebruari 2004 sejumlah peneliti di Korea telah mengumumkan pembuatan stem cell manusia pertama dengan cara transplantasi sel somatik. Walaupun pernyataan ini kemudian ditarik kembali dengan alasan manipulasi data atau perilaku tidak etis para penelitinya, hal ini telah mendorong para peneliti untuk menggiatkan penelitian sel punca dan pengkolnan embrio guna pemakaian dalam pengobatan penyakit-penyakit degeneratif. Penelitian dengan menggunakan embrio dan pengklonan embrio telah menyulut kontroversi dan menjadi bahan perdebatan dibanyak negara, seperti Inggris, Amerika Serikat, Swedia dan sebagainya. Uraian dibawah ini akan membahas tentang pengertian, aspek biomedik dan potensi penggunaannya di klinik serta masalah etik yang membayanginya DEFINISI Sel Punca atau stem cell adalah sel yang tidak/belum terspesialisasi dan mempunyai kemampuan/potensi untuk berkembang menjadi berbagai jenis sel-sel yang spesifik yang membentuk berbagai jaringan tubuh. Gambar-1 Sifat/karakter sel punca yaitu differentiate dan self regenerate/renew Sel Punca mempunyai 2 sifat yang khas yaitu 1. Differentiate yaitu kemampuan untuk berdifferensiasi menjadi sel lain. Sel Punca mampu berkembang menjadi berbagai jenis sel yang khas (spesifik) misalnya sel saraf, sel otot jantung, sel otot rangka, sel pankreas dan lain-lain 2. Self regenerate/self renew yaitu kemampuan untuk memperbaharui atau meregenerasi dirinya sendiri. Stem cells mampu membuat salinan sel yang persis sama dengan dirinya melalui pembelahan sel. Berdasarkan pada kemampuannya untuk berdifferensiasi sel punca dikelompokkan menjadi 1. Totipoten yaitu sel punca yang dapat berdifferensiasi menjadi semua jenis sel. Yang termasuk dalam sel punca totipoten adalah zigot dan morula. Sel-sel ini merupakan sel embrionik awal yang mempunyai kemampuan untuk membentuk berbagai jenis sel termasuk sel-sel yang menyusun plasenta dan tali pusat. Karenanya sel punca kelompok ini mempunyai kemampuan untuk membentuk satu individu yang utuh. Gambar-2 Sel Punca totipoten dan pluripoten 2. Pluripoten yaitu sel punca yang dapat berdifferensiasi menjadi 3 lapisan germinal (ektoderm, mesoderm, dan endoderm) tetapi tidak dapat menjadi jaringan ekstraembrionik seperti plasenta dan tali pusat. Yang termasuk sel punca pluripoten adalah sel punca embrionik (embryonic stem cells). 3. Multipoten yaitu sel punca yang dapat berdifferensiasi menjadi berbagai jenis sel misalnya sel punca hemopoetik (hemopoetic stem cells) yang terdapat pada sumsum tulang yang mempunyai kemampuan untuk berdifferensiasi menjadi berbagai jenis sel yang terdapat di dalam darah seperti eritrosit, lekosit dan trombosit. Contoh lainnya adalah sel punca saraf (neural stem cells) yang mempunyai kemampuan berdifferensiasi menjadi sel saraf dan sel glia. 4. Unipotent yaitu sel punca yang hanya dapat berdifferensiasi menjadi 1 jenis sel. Berbeda dengan non sel punca, sel punca mempunyai sifat masih dapat memperbaharui atau meregenerasi diri (self-regenerate/self renew) Contohnya erythroid progenitor cells hanya mampu berdifferensiasi menjadi sel darah merah. Gambar-3 Multipotent dan unipotent stem cells pada sumsum tulang ASPEK BIOMEDIK SEL PUNCA EMBRIONIK (EMBRYONIC STEM CELLS) Sel punca embrionik (embryonic stem cells) adalah sel yang diambil dari inner cell mass (suatu kumpulan sel yang terletak di satu sisi blastokista) embrio berumur 5 hari dan terdiri dari 100 sel. Sel ini mempunyai sifat dapat berkembang biak secara terus menerus dalam media kultur optimal dan dalam keadaan tertentu dapat diarahkan untuk berdifferensiasi menjadi berbagai sel yang terdifferensiasi seperti sel jantung, sel kulit, neuron, hepatosit dan sebagainya, sehingga dapat dipakai untuk transplantasi jaringan yang rusak. Gambar -4 Embryonic Stem Cells Inner cell mass ini mempunyai kemampuan untuk menjadi berbagai jaringan embrio dan tubuh kecuali membentuk plasenta. Inner cell mass ini disebut sel pluripotent karena dapat berkembang lebih lanjut menjadi berbagai jaringan dan organ tubuh. Secara alami sel pluripotent yang telah berkembang dan melakukan spesialisasi dikenal sebagai sel multipoten dan merupakan sel punca dewasa. Sel punca dewasa ini dapat berkembang menjadi berbagai sel dan jaringan. Tantangan bagi peneliti sebenarnya adalah cara memanipulasi sel punca dewasa ini sehingga berkembang menjadi sel atau produk yang diinginkan yang dapat digunakan untuk pengobatan. Sel punca embrionik (Embryonic Stem Cell) mempunyai sifat sebagai berikut 1. pluripoten, artinya sel punca ini mempunyai kemampuan berdifferensiasi menjadi sel-sel yang merupakan turunan dari 3 lapis germinal, tetapi tidak dapat membentuk membran embrio (tali pusat dan plasenta) 2. immortal artinya dapat berumur panjang sehingga dapat memperbanyak diri ratusan kali pada media kultur. Mereka merupakan sumber sel-sel yang belum berdifferensiasi. Sel punca embrionik dulu dipikirkan dapat memperbanyak diri sendiri secara tak terbatas, tetapi kini diketahui bahwa usia dan perbanyakan diri sendiri sel-sel stem juga ada batasnya. Hal ini disebabkan karena terjadinya mutasi pada gen-gen pada sel stem yang diakibatkan karena pengaruh nutrisi dalam medium kultur. 3. mempunyai karyotipe yang normal 4. dapat bersifat tumorigenik artinya setiap kontaminasi dengan sel yang tak berdifferensiasi dapat menimbulkan kanker 5. selalu bersifat allogenik sehingga berpotensi menimbulkan terjadinya rejeksi imunitas. Untuk mencegah terjadinya reaksi penolakan jaringan dapat digunakan metoda somatic cell nuclear transfer atau terapi kloning. Gambar-5 Metoda Somatic Cell Nuclear Transfer Therapeutic cloning atau disebut Somatic Cell Nuclear Transfer (SCNT) adalah suatu teknik yang bertujuan untuk menghindari resiko penolakan atau rejeksi. Pada teknik ini inti sel telur donor dikeluarkan dan diganti dengan inti sel resipien. Sel yang telah dimanipulasi ini kemudian akan membelah diri dan setelah menjadi blastokista maka inner cell massnya akan diambil sebagai embryonic stem cells. Stem cells ini kemudian akan dimasukkan kembali kedalam tubuh resipien dan stem cells ini kemudian akan berdifferensiasi menjadi sel organ (sel beta pankreas, sel otot jantung dan lain-lain). Tanpa reaksi penolakan karena sel tersebut mengandung materi genetik resipien. Gambar-6 Terapi Kloning (Therapeutic Cloning) APPLIKASI / PENGGUNAAN KULTUR STEM CELLS Stem cells dapat digunakan untuk keperluan baik dalam bidang riset maupun pengobatan. Adapun penggunaan kultur stem cells adalah sebagai berikut 1. Terapi gen Stem cells khususnya hematopoetic stem cells digunakan sebagai pembawa transgen kedalam tubuh pasien dan selanjutnya dilacak apakah jejaknya apakah stem cells ini berhasil mengekspresikan gen tertentu dalam tubuh pasien. Adanya sifat self renewing pada stem cell menyebabkan pemberian stem cells yang mengandung transgen tidak perlu dilakukan berulang-ulang. Selain itu hematopoetic stem cells juga dapat berdifferensiasi menjadi bermacam-macam sel sehingga transgen tersebut dapat menetap diberbagai macam sel. 2. Penelitian untuk mempelajari proses-proses biologis yang terjadi pada organisma termasuk perkembangan organisma dan perkembangan kanker 3. Penelitian untuk menemukan dan mengembangkan obat-obat baru terutama untuk mengetahui efek obat terhadap berbagai jaringan 4. Terapi sel (cell based therapy) Stem cell dapat hidup diluar tubuh manusia, misalnya di cawan Petri. Sifat ini dapat digunakan untuk melakukan manipulasi pada stem cells yang akan ditransplantasikan ke dalam organ tubuh untuk menangani penyakit-penyakit tertentu tanpa mengganggu organ tubuh. Gambar-7 Berbagai peran Stem Cells PENGGUNAAN STEM CELLS DALAM PENGOBATAN PENYAKIT Para ahli saat ini sedang giat melakukan berbagai penelitian untuk menggunakan stem cell dalam mengobati berbagai penyakit. Penggunaan stem cells untuk mengobati penyakit dikenal sebagai Cell Based Therapy. Prinsip terapi adalah dengan melakukan transplantasi stem cells pada organ yang rusak. Tujuan dari transplantasi stem cells ini adalah 1. Mendapatkan pertumbuhan dan perkembangan sel-sel baru yang sehat pada jaringan atau organ tubuh pasien 2. Menggantikan sel-sel spesifik yang rusak akibat penyakit atau cidera tertentu dengan sel-sel baru yang ditranspalantasikan. Sel stem embryonic sangat plastik dan mempunyai kemampuan untuk dikembangkan menjadi berbagai macam jaringan sel seperti neuron, kardiomiosit, osteoblast, fibroblast, sel-sel darah dan sebagainya, sehingga dapat dipakai untuk menggantikan jaringan yang rusak. Sel stem dewasa juga dapat digunakan untuk mengobati berbagai penyakit degeneratif, tetapi kemampuan plastisitasnya sudah berkurang. Keuntungan dari penggunaan sel stem dewasa yaitu tidak atau kurang menimbulkan masalah dan kontroversi etika. Penggunaan sel punca embrionik untuk mengobati cidera pada medula spinalis (spinal cord) Cidera pada medula spinalis disertai demielinisasi menyebabkan hilangnya fungsi neuron. Sel punca dapat mengembalikan fungsi yang hilang dengan cara melakukan remielinisasi . Percobaan dengan sel punca embrionik tikus dapat menghasilkan oligodendrosit yang kemudian dapat menyebabkan remielinisasi akson yang rusak Penggunaan sel punca pada penyakit stroke Pada penyakit stroke dicoba untuk menggunakan sel punca mesenkim (mesenchymal stem cells (MSC) dari sumsum tulang autolog. Penelitian ini didasarkan pada hasil penelitian yang telah dilakukan sebelumnya. Mesenchymal stem cells diperoleh dari aspirasi sumsum tulang. Setelah disuntikkan perifer MSC akan melintas sawar darah otak pada daerah otak yang rusak. Pemberian MSC intravenous akan mengurangi terjadinya apoptosis dan menyebabkan proliferasi sel endogen setelah terjadinya stroke Penggunaan sel punca pada infark miokardium Bartinek telah melakukan intracoronary infusion bone marrow stem cells otolog pada 22 pasien dengan AMI dan mendapatkan hasil yang baik. Penelitian terkini menunjukkan bukti awal bahwa adult stem cells dan embryonic stem cells dapat menggantikan sel otot jantung yang rusak dan memberikan pembuluh darah baru. Strauer et al. mencangkok mononuclear bone marrow cell autolog ke dalam arteri yang menimbulkan infark pada saat PTCA 6 hari setelah infark miokard. Sepuluh pasien yang diberi stem cells area infarkya menjadi lebih kecil dan indeks volume stoke, left ventricular end systolic volume, kontraktilitas area infark dan perfusi miokard menunjukkan perbaikan dibandingkan dengan kelompok kontrol. Bioetika Pada Penelitian Stem Cells Manfaat yang diperoleh dari penggunaan sel punca embrionik (embryonic stem cell) dalam bidang kedokteran amat besar, namun sumber sel punca embrionik ini merupakan masalah etika yang perlu mendapat perhatian. Berkembangnya penelitian stem cell dan penggunaan stem cell dalam upaya untuk mengobati penyakit pada manusia akan mengakibatkan timbulnya masalah dalam hal etik. Hal utama terkait dengan masalah etik adalah sumber stem cell tersebut. Berbagai masalah etika yang perlu dipikirkan adalah 1. Apakah penelitian embrio manusia secara moral dapat dipertanggung jawabkan? 2. Apakah penelitian embrio yang menyebabkan kematian embrio merupakan pelanggaran terhadap hak azasi manusia (HAM) dan berkurangnya penghormatan terhadap mahluk hidup? 3. Apakah penyalah gunaan dapat diketahui dan dikendalikan? 4. Apakah penggunaan embrio sisa proses bayi tabung pada penelitian diperbolehkan? 5. Apakah penelitian khusus membuat embrio untk digunakan diperbolehkan? Isu bioetika utama dalam penelitian dan penggunaan stem cell adalah penggunaan stem cell embrio terutama tentang sumber sel tersebut yaitu embrio. Sumber embrio adalah hasil abortus, zigot sisa IVF dan hasil pengklonan. Pengklonan embrio manusia untuk memperoleh stem cell merupakan isu yang sangat menimbulkan kontroversi. Hal ini terkait dengan isu ”awal kehidupan” dan penghormatan terhadap kehidupan. Pengklonan embrio manusia untuk memperoleh stem cell menimbulkan kontroversi karena berhubungan dengan pengklonan manusia yang ditentang oleh semua agama. Dalam proses pemanenan stem cell embrio terjadi kerusakan pada embrio dan menyebabkan embrio tersebut mati. Adanya anggapan bahwa embrio berstatus sama dengan manusia menyebabkan hal tersebut tidak dapat diterima Perdebatan yang cukup ramai adalah mengenai status moral embrio, apakah embrio harus diperlakukan sebagai manusia atau sebagai sesuatu yang berpotensi untuk menjadi manusia atau sebagai jaringan hidup tubuh lainnya. Lebih jauh lagi apakah embrio yang berkembang dianggap sebagai mahluk hidup. Penggunaan stem cell yang berasal dari surplus zigot pembuatan bayi tabung sendiri. juga menimbulkan kontroversi. Pendapat yang moderat mengatakan ketimbang surplus zigot itu dibuang, sebaiknya dipakai saja untuk penelitian. Sebaliknya ada juga yang berpendapat bahwa sisa itu harus dipelihara hingga zigot itu mati. KEPUSTAKAAN 1. McNeish, J. (2004) Embryonic Stem Cells in Drug Discovery Nat. Rev. Drug Discov. 3, 70-80 2. Davila, J.C., Cezar, G.G., Thiede, M., Strom, S., Miki, T., Trosko J. (2004) Use and Application of Stem Cells in Toxicology. Toxicol. Sci. 79, 214-223 3. The stem Cell- Stem cell information- The Official national Institute of Health Resource for Stem Cell Research . 4. Anatomy 101: Stem cell-Reeve Irvine Research Center- http/ www.reeve.uci.edu/anatomy/stem cells.php. 5. Sell, S. (2004) Stem cells. Stem Cell Handbook ed. by Sell, S. 1-18. 6. FOXNews.com - New Stem-Cell Procedure Doesn't Harm Embryos, Company Claims - Biology | Astronomy | Chemistry | Physics 7. Therapeutic use of cell nuclear replacement: Therapeutic cloning-Research in focus- MRC (Medical Research Council) 8. For review: Floss,T., Wurst, W. (2002) Functional Genomics by Gene-trapping in ES cells. Embryonic Stem Cells Methods and Protocols ed. by Turksen, K. 347379 9. What are stem eclls? – CSA guide to discovery – http://www.csa.com/discovery guide/stem cell//overview.php 10. Liu S, Qu Y, Stewart TJ et al. Embryonic stem cells differentiate into oligodendrocyts and myelinated in culture and after spinal cord transplantation. PNAS 2000: 97(11):6126-6131 11. Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery Neurology 2002;59:514 –523 12. Zhao LR, Duan WM, Reyes M, et al. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. ExpNeurol 2002;174:11–20) 13. Bartinek J, Vanderheyden M, Vandekerchove B et al., Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction. Circulation 2005; 112 (9 suppl): 78-83 14. Stem cells transplantation in myocard infarction: A status report- Ann Intern. Med. 2004 May: 140(9): 729-737 15. Setiawan B, Aplikasi teraupetik sel stem embrionik pada berbagai penyakit degeneratif. Cermin Dunia Kedokteran 2006; 153: 5-8 16. Tadjudin MK, Aspek bioetika penelitin stem cell. Cermin Dunia Kedokteran 2006; 153: 9-12. 17. Kaligis RWM, Aplikasi terapi stem cell pada infark miokard akut. Cermin Dunia Kedokteran 2006; 153: 13 18. Reksodiputro AH, Stem cell therapy in hematologic malignancies. Cermin Dunia Kedoketran 2006; 153: 14-15 19. Setyopranoto I, Application of stem cell therapy in Parkinson Disease. Cermin Dunia Kedokteran 2006; 153: 16 20. Islam MS, Terapi sel stem pada cedera medulla spinalis. Cermin Dunia Kedokteran 2006; 153: 17-19 21. Ibrahim N, Aplikasi terapi stem cell pada luka bakar. Cermin Dunia Kedoketran 2006; 153: 20 22. Saputra V, Dasar-dasar stem cell dan potensi apilkasinya dalam ilmu kedokteran. Cermin Dunia Kedoketran 2006; 153: 21-25 23. Prayogo R, Wijaya MT, Kultur dan potensi stem cells dari darah tali pusat. Cermin Dunia Kedoketran 2006; 153: 26-28 ES cells from mouse embryos have been cultured since the 1980s by various groups of researchers working independently.10 These pioneers established murine embryonic stem cells lines that could differentiate into several different cell types.11 ES cell lines have been established from other mammals (hamsters, rats, pigs, and cows). Thompson and colleagues at the University of Wisconsin reported isolation of primate ES cells in 1995 and human ES cells in 1998.12 B. Research and Clinical Applications of Cultured Stem Cells What are the uses of Cultured Stem cells? The most prominent is cell therapy for treating conditions such as spinal cord injuries and for curing disease. Stem cells are used to investigate questions to further basic and clinical research. Here are the major applications to date: 1. Functional Genomic studies In 1986, Gossler et al. reported using mouse ES cells to produce transgenic animals.24 Soon after, two landmark papers in the field of mouse genetics demonstrated the ability to manipulate a specific gene of ES cells.25 Combining these techniques, a specific gene can be introduced into ES cells to produce transgenic mice. This gene can be transmitted to their offspring through the germline. Today these techniques enable the study of the function of mammalian genes and proteins in the mouse (through introducing human histocompatibility genes into mice).26 2. Study of biological processes Studies of biological processes, namely development of the organism and progress of cancer, are facilitated by the ability to trace stem cell fate. The spleen colony assay developed by Till and McCulloch is an example study of the development of blood cells. In this method single cells were injected into heavily irradiated mice so that all the hematopoietic cells in these mice originated from the original colony. Studies of this nature helped decipher the clonal origin of cancer, 3. Drug discovery and development The combination of isolation and purification of mouse ES cells and genetic engineering techniques has led to the use of murine ES cells in drug discovery. With the sequencing of the human genome many potential targets of new drugs have been identified. Studies using human ES may follow those of murine ES cells.27 Interest in using stem cells as models for toxicology has also grown recently.28 4. Cell-based therapy Cultured ES cells spontaneously form embryoid bodies containing different cell types from all three germ layers. The desired cells are isolated and cultured and the differentiated cells are then used for therapy. ES cells have been induced to differentiate into neurons, cardiomyocytes and endoderm cells. 1. For ethical issues and stem cell research refer to http://stemcells.nih.gov/info/ethics.asp; http://athome.harvard.edu/programs/psc/index.html; http://www.aaas.org/spp/sfrl/projects/stem/main.htm Ethical Issues Associated with Pluripotent Stem Cells. Human Embryonic Stem Cells (2003) ed. by Chiu A.Y., Rao, M.S, 3-25. 2. Sell, S. (2004) Stem cells. Stem Cell Handbook ed. by Sell, S. 1-18. 3. http://stemcells.nih.gov/info/scireport/chapter4.asp 4. Forbes, S.J., Vig, P., Poulsom, R., Wright, N.A., Alison, M.R. (2002) Adult Stem Cell Plasticity: New Pathways of Tissue Regeneration become Visible. Clin. Sci. 103, 355-369. 5. Asahara T., Isner, J.M. (2004) Endothelial Progenitor Cells. Stem Cell Handbook ed. by Sell, S. 221-227. 6. Lindblad, W.J. (2004) Stem cells in Dermal Wound Healing. Stem Cell Handbook ed. by Sell, S. 101-105. 7. McCulloch, E.A. (2004) Normal and Leukemic Hematopietic Stem cells and Lineages. Stem Cell Handbook ed. by Sell, S. 119-131. 8. Tsai, R.Y.L. (2004) A Molecular View of Stem Cell and Cancer Cell Self-renewal. Intl. J. Biochem. Cell Biol. 36, 684-694. 9. Cai, J., Weiss M.L., Rao, M.S. (2004) In Search of "stemness". Exp. Hematol. 32, 585-598. 10. Roach, M.L., McNeish, J.D. (2002) Methods for the Isolation and Maintenance of Murine Embryonic Stem Cells. Embryonic Stem Cells Methods and Protocols ed. by Turksen K. 1-16. back to article 11. Evans, M.J., Kaufman, M.H. (1981) Establishment in Culture of Pluripotenial Cells from Mouse Embryos. Nature 292, 154-156; Axelrod, H.R. (1984) Embryonic Stem Cell Lines Derived from Blastocysts by a Simplified Technique. Dev. Biol. 101, 225-228; Wobus, A.M., Holzhausen H., Jakel, P., Schneich, J. (1984) Characterization of a Pluripotent Stem Cell Line Derived from a Mouse Embryo. Exp. Cell Res. 152, 212-219; Doetschman, T.C. Eistattaer, H., Katz, M., Schmidt, W., and Kemler, R. (1985) The in vitro development of Blastocyst Derived Embryonic Stem Cell Lines: formation of Yolk Sac, Blood Islands and Myocardium. J. Embryol. Exp. Morphol. 87, 27-45. 12. Thompson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A., Hearn, J.P. (1995) Isolation of a Primate Embryonic Stem Cell Line. Proc. Natl. Acad. Sci. USA 86, 7844-7848; Thomson, J.A, Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshal, V.S., Jones, J.M. (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282, 1145-1147. 13. Amit, M., Segev, H., Manor, D., Itskovitz-Eldor, J. (2003) Subcloning and Alternative Methods for the Derivation and Culture of Human Embryonic Stem Cells. Human Embyronic Stem Cells ed. by Chiu, M., Rao, M.S. 127-141. 14. Carpenter, M.K., Xu, C., Daigh, C.A., Antosiewicz, J.E., Thomson, J.A. (2003) Protocols for the Isolation and Maintenance of Human Embryonic Stem Cells. Human Embyronic Stem Cells ed. by Chiu, M., Rao, M.S. 15. Drukker M., Benvenisty, N. (2003) Genetic Manipulation of Human Embryonic Stem Cells. Human Embryonic Stem Cells ed. by Chiu, A.Y., Rao, M.S. 265-284. back to article 16. Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G. R., Gearhart J.D., (1998) Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ CellS. Proc. Natl. Acad. Sci.USA 95, 13726-13731. 17. Doyonnas, R., Blau, H.M. (2004) What is the Future of Stem Cell Research? Stem Cell Handbook ed. by Sell, S. 491-499. 18. Draper, J.S. Moore, H., Andrews, P.W. (2003) Embryonal Carcinoma Cells. Human Embryonic Stem Cells ed. Chiu, A. Y., Rao, M.S. 63-87. 19. Adult Stem Cells ed. Turksen, K. (2004) For reviews of hematopoietic stem cells: http://stemcells.nih.gov/info/scireport/chapter5.asp; http://www.stemcell.com/technical/Hema%20SC%20MiniReview.pdf; for mesenchymal stem cells: http://www.stemcell.com/technical/MSC %20MiniReview.pdf; for neural stem cells: http://www.stemcell.com/technical/Neurocult%20MiniReview.pdf 20. Nosrat, I.V., Smith, C. A., Mullally, P., Olson, L., Nosrat C.A. (2004) Dental Pulp Cells Provide Neurotrophic Support for Dopaminergic Neurons and Differentiate into Neurons in vitro; implications for Tissue Engineering and Repair in the Nervous System. Eur. J. of Neurosci. 19, 2388-2398. back to article 21. Shen, C-N., Horb, M.E., Slack, J.M.W., Tosh,D. (2003) Transdifferentiation of Pancreas to Liver. Mech. Dev.120, 107-116. 22. Priller, J. (2004) From Marrow to Brain. Adult Stem Cells ed. by Turksen, K. 215233. 23. de Wynter, E.A. (2003) What is the future of Cord blood stem cells? Cytotech. 41, 133-138. 24. Gossler, A., Doetschman, T.C., Eistattaer, H., Katz, M., Schmidt, W., Kemler, R. (1986) Transgenesis by means of Blastocyst Derived Embryonic Stem Cell Lines. Proc. Natl. Acad. Sci. USA 83, 9065-9069. 25. Thomas, K.R., Capecchi, M.R. (1987) Site-directed Mutagenesis by Gene Targeting in Mouse Embryo-derived Stem Cells. Cell 51, 503-512.; Koller, B.H., Hageman, L.J., Doetschman, T.C., Hagaman, J.R., Huang, S., Williams, P.J., et. al. (1989) Proc. Natl. Acad. Sci. USA 86, 8924-8931. back to article 26. For review: Floss,T., Wurst, W. (2002) Functional Genomics by Gene-trapping in ES cells. Embryonic Stem Cells Methods and Protocols ed. by Turksen, K. 347379. 27. McNeish, J. (2004) Embryonic Stem Cells in Drug Discovery Nat. Rev. Drug Discov. 3, 70-80. 28. Davila, J.C., Cezar, G.G., Thiede, M., Strom, S., Miki, T., Trosko J. (2004) Use and Application of Stem Cells in Toxicology. Toxicol. Sci. 79, 214-223. 29. Till, J.E., McCulloch, E.A. (1961) A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells. Radiat. Res. 14, 2213-222. 30. Thomas, E.D. (1999) Bone Marrow Transplantation: a Review. Semin. Hematol. 36, 95-103. back to article 31. Barker, R.A., Jain, M., Armstrong, R.J.E., Caldwell, M.A. (2003) Stem Cells and Neurological Disease. J. Neurol. Neurosurg. Psychiat. 74, 553-557. 32. http://www.who.int/cardiovascular_diseases/resources/atlas/en/ 33. Jackson, K.A., Goodell, M.A. (2004) Generation and Stem Cell Repair of Cardiac Tissue. Stem Cell Handbook, edited by Sell, S. 259-266. 34. Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G., Huber, I., Satin, J., Itskovitz-Eldor, J., Gepstein, L. (2004) Electromechanical Integration of Cardiomyocytes Derived from Human Embryonic Stem Cells . Nature Biotechnol. 22, 1282-1289. 35. Fraser, J.K., Schreiber, R.E., Zuk, P.A., Hedrick, M.H. (2004) Adult Stem Cell Therapy for the Heart. Intl. J. Biochem. Cell Biol. 36, 658-666. back to article 36. Cohen, S., Leor, J. (2004) Rebuilding Broken Hearts. Scientific American Nov. 2004, 45-51. 37. http://stemcells.nih.gov/info/scireport/chapter7.asp; Street, C.N., Sipione, S., Helms, L., Binette, T., Rajotte, R.V., Bleackley, R.C., Korbutt, G.S. (2004) Stem Cell-based Approaches to Solving the Problem of Tissue Supply for Islet Transplantation in Type I Diabetes. Intl. J. Biochem. Cell Biol. 36, 667-683. 38. Bouwens, L. (2004) Islet Cells. Stem Cell Handbook ed. by Sell, S. 429-438. 39. Seaberg, R.M., Smukler, S.R., Kieeffer, T.J., Enikolopov, G., Asghar, Z., Wheeler M.B., Korbutt, G., van der Kooy, D. (2004) Clonal Identification of Multipotent Precursors from Adult Mouse Pancreas that Generate Neural and Pancreatic Lineages. Nat. Biotechnol. 22, 1115-1124.; SeNakajima-Nagata, N., Sakurai, T., Mitaka, T., Katakai, T., Yamaot, E., Miyazaki, J., Tabata, Y., Sugai, M., Shimzu, A.. (2004) In vitro Induction of Adult Hepatic Progenitor Cells into Insulinproducing Cells. Biochem. Biophys. Res. Commun. 318, 625-630. 40. http://www.parkinson.org/site/pp.asp?c=9dJFJLPwB&b=71125 back to article 41. http://stemcells.nih.gov/info/scireport/chapter8.asp 42. Baier, P.C., Schindehutte HJ., Thinane, K., Flugge G., Fuchs, E., Mansouri, A., Paulus, W., Gruss, P.,Trenwalder, C.(2004) Behavioral Changes in Unilaterally 6Hydroxy-Dopamine Lesioned Rats after Transplantation of Differentiated Mouse Embryonic Stem Cells without Morphological Integration. Stem Cells 22, 396404. 43. Lindvall O., Bjorklund, A. (2004) Cell Therapy in Parkinson's Disease. NeuroRx. 1, 382-393. 44. Zheng, X., Cai, J., Chen, J., Luo, Y., Zhi-Bing Y., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., Chen, G-J., Rao, M.S., Freed. W.J. (2004) Dopaminergic Differentiation of Human Embryonic Stem Cells. Stem Cells 22, 925-940; Wilmut, I., Paterson, L.A. (2004) Stem cells and Cloning. Stem Cell Handbook ed. by Sell, S. 75-80. Barker, R.A., Widner, H. (2004). Immune Problems in the Central Nervous System Cell Therapy. NeuroRx. 1, 472-481. Embryonic stem cells (ES cells) were first derived from mouse embryos in 1981 by Martin Evans and Matthew Kaufman and independently by Gail R. Martin. Gail R. Martin is credited with coining the term 'Embryonic Stem Cell'.[3][4] A breakthrough in human embryonic stem cell research came in November 1998 when a group led by James Thomson at the University of Wisconsin-Madison first developed a technique to isolate and grow the cells when derived from human blastocysts.[5] [edit] Potential method for new cell line derivation On August 23, 2006, the online edition of Nature scientific journal published a letter by Dr. Robert Lanza (medical director of Advanced Cell Technology in Worcester, MA) stating that his team had found a way to extract embryonic stem cells without destroying the actual embryo.[9] This technical achievement would potentially enable scientists to work with new lines of embryonic stem cells derived using public funding. Federal funding is currently limited to research using embryonic stem cell lines derived prior to August 2001. See also: Induced pluripotent stem cell Professor Yamanaka had a recent breakthrough[10] in which the skin cells of laboratory mice were genetically manipulated back to their embryonic state. This work was confirmed by two other groups, demonstrating that the addition of just 4 genes (Oct3/4, Sox2, Klf4, and c-Myc) could reprogram mouse skin cells into embryonic stem like cells. The ability to reproduce such findings are very important in science and the stem cell field, especially after Hwang Woo-Suk from Korea fabricated data, claiming to have generated human ES cells from cloned embryos. These cells produced by Yamanaka as well as the other laboratories demonstrated all the hallmarks of embryonic stem cells including the ability to form chimeric mice and contribute to the germ-line. One issue with this work is that the mice generated from these ES lines were prone to develop cancer due to the use of Myc, which is a known oncogene. On 20th of November, 2007, two research teams, one of which was headed by Professor Yamanaka and the other by James Thomson[11] announced a similar breakthrough with ordinary human skin cells that were transformed into batches of cells that look and act like embryonic stem cells. This may enable the generation of patient specific ES cell lines that could potentially be used for cell replacement therapies. In addition, this will allow the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study those diseases. While this work is a huge accomplishment for science, there is still much work to be done before this technology can be used for the treatments of disease. First, the genes used to reprogram the skin cells into ES-like cells were added by the use of retroviruses that can cause mutations and lead to the risk of possible cancers, although recent research by professor Yamanaka's research group has made advances in avoiding this particular problem.[12] In addition, as shown with the mouse work, one of the genes used to reprogram, Myc, can also cause cancer. The group led by Thomson did not use Myc to reprogram and may not have this difficulty. Future work is aimed at attempting to reprogram without permanent genetic manipulation of the cells with viruses. This could be accomplished by either small molecules or other methodologies to express these reprogramming genes. However, as a first indication that the induced pluripotent stem (iPS) cell technology can in rapid succession lead to new cures, it was used by a research team headed by Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, to cure mice of sickle cell anemia, as reported by Science journal's online edition on 6th of December.[13] On January 16, 2008, a California based company, Stemagen, announced that they had created the first mature cloned human embryos from single skin cells taken from adults. These embryos can be harvested for patient matching embryonic stem cells.[14] 1. ^ Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (August 25, 2006). "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors". Cell. 2. ^ Andrews P, Matin M, Bahrami A, Damjanov I, Gokhale P, Draper J (2005). "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin.". Biochem Soc Trans 33 (Pt 6): 1526-30. PMID 16246161. 3. ^ Evans M, Kaufman M (1981). "Establishment in culture of pluripotential cells from mouse embryos.". Nature 292 (5819): 154-6. doi:10.1038/292154a0. PMID 7242681. 4. ^ Martin G (1981). "Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.". Proc Natl Acad Sci U S A 78 (12): 7634-8. doi:10.1073/pnas.78.12.7634. PMID 6950406. 5. ^ Thomson J, Itskovitz-Eldor J, Shapiro S, Waknitz M, Swiergiel J, Marshall V, Jones J (1998). "Embryonic stem cell lines derived from human blastocysts.". Science 282 (5391): 1145-7. doi:10.1126/science.282.5391.1145. PMID 9804556. 6. ^ "Derivation of embryonic germ cells and male gametes from embryonic stem cells" (January 8, 2004). Nature 427: 148-154. doi:10.1038/nature02247. 7. ^ Access to articles : Nature Medicine 8. ^ Lancet Medical Journal 9. ^ Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. (2006). "Human embryonic stem cell lines derived from single blastomeres.". Nature 444 (7118): 481-5. PMID 16929302. 10. ^ "Human stem cells may be produced without embryos ‘within months’", Zangani, July 17, 2007. 11. ^ "Embryonic stem cells made without embryos", Reuters, November 21, 2007. 12. ^ "Researchers get closer to safe stem cell treatments", AFP, February 14, 2008. 13. ^ Rick Weiss. "Scientists Cure Mice Of Sickle Cell Using Stem Cell Technique: New Approach Is From Skin, Not Embryos", Washington Post, December 7, 2007, pp. A02. 14. ^ Helen Briggs. "US team makes embryo clone of men", BBC, January 17, 2008, pp. A01. Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos.[6] A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF).[7] Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2).[8] Without optimal culture conditions or genetic manipulation,[9] embryonic stem cells will rapidly differentiate. A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and SOX2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency.[10] The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.[11] The signals that lead to reprogramming of cells to an embryonic-like state are also being investigated. These signal pathways include several transcription factors including the oncogene c-Myc. Initial studies indicate that transformation of mice cells with a combination of these anti-differentiation signals can reverse differentiation and may allow adult cells to become pluripotent.[24] However, the need to transform these cells with an oncogene may prevent the use of this approach in therapy. There exists a widespread controversy over human embryonic stem cell research that emanates from the techniques used in the creation and usage of stem cells. Human embryonic stem cell research is controversial because, with the present state of technology, starting a stem cell line requires the destruction of a human embryo and/or therapeutic cloning. However, recently, it has been shown in principle that embryonic stem cell lines can be generated using a single-cell biopsy similar to that used in preimplantation genetic diagnosis that may allow stem cell creation without embryonic destruction.[29] It is not the entire field of stem cell research, but the specific field of human embryonic stem cell research that is at the centre of an ethical debate. Opponents of the research argue that embryonic stem cell technologies are a slippery slope to reproductive cloning and can fundamentally devalue human life. Those in the pro-life movement argue that a human embryo is a human life and is therefore entitled to protection. Contrarily, supporters of embryonic stem cell research argue that such research should be pursued because the resultant treatments could have significant medical potential. It is also noted that excess embryos created for in vitro fertilisation could be donated with consent and used for the research. The ensuing debate has prompted authorities around the world to seek regulatory frameworks and highlighted the fact that stem cell research represents a social and ethical challenge. Key stem cell research events 1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; their reports contradict Cajal's "no new neurons" dogma and are largely ignored. 1963 - McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow. 1968 - Bone marrow transplant between two siblings successfully treats SCID. 1978 - Haematopoietic stem cells are discovered in human cord blood. 1981 - Mouse embryonic stem cells are derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell". 1992 - Neural stem cells are cultured in vitro as neurospheres. 1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells. 1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of Wisconsin-Madison. 2000s - Several reports of adult stem cell plasticity are published. 2001 - Scientists at Advanced Cell Technology clone first early (four- to six-cell stage) human embryos for the purpose of generating embryonic stem cells.[30] 2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in children's primary teeth.[31] 2004-2005 - Korean researcher Hwang Woo-Suk claims to have created several human embryonic stem cell lines from unfertilised human oocytes. The lines were later shown to be fabricated. 2005 - Researchers at Kingston University in England claim to have discovered a third category of stem cell, dubbed cord-blood-derived embryonic-like stem cells (CBEs), derived from umbilical cord blood. The group claims these cells are able to differentiate into more types of tissue than adult stem cells. August 2006 - Rat Induced pluripotent stem cells: the journal Cell publishes Kazutoshi Takahashi and Shinya Yamanaka, "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors". October 2006 - Scientists in England create the first ever artificial liver cells using umbilical cord blood stem cells.[32][33] January 2007 - Scientists at Wake Forest University led by Dr. Anthony Atala and Harvard University report discovery of a new type of stem cell in amniotic fluid.[5] This may potentially provide an alternative to embryonic stem cells for use in research and therapy.[34] June 2007 - Research reported by three different groups shows that normal skin cells can be reprogrammed to an embryonic state in mice.[35] In the same month, scientist Shoukhrat Mitalipov reports the first successful creation of a primate stem cell line through somatic cell nuclear transfer[36] October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel Prize for Physiology or Medicine for their work on embryonic stem cells from mice using gene targeting strategies producing genetically engineered mice (known as knockout mice) for gene research.[37] November 2007 - Human Induced pluripotent stem cells: Two similar papers released by their respective journals prior to formal publication: in Cell by Kazutoshi Takahashi and Shinya Yamanaka, "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors", and in Science by Junying Yu, et al., from the research group of James Thomson, "Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells": pluripotent stem cells generated from mature human fibroblasts. It is possible now to produce a stem cell from almost any other human cell instead of using embryos as needed previously, albeit the risk of tumorigenesis due to c-myc and retroviral gene transfer remains to be determined. January 2008 - Human embryonic stem cell lines were generated without destruction of the embryo[38] January 2008 - Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts[39] February 2008 - Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach: these iPS cells seem to be more similar to embryonic stem cells than the previous developed iPS cells and not tumorigenic, moreover genes that are required for iPS cells do not need to be inserted into specific sites, which encourages the development of non-viral reprogramming techniques. [40][41] ^ Becker AJ, McCulloch EA, Till JE (1963). "Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells". 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