Adım 1: 293 lentivirüs üretim hücreleri ve plaka çözülme. <ol><li> 9 ml, 15 ml tüp DMEM + orta hazırlayın. </li><li> Dondurulmuş 293 hisse senetlerinin bir şişe sıvı nitrojen tankı çıkarın ve (hepsi değil) en hücreleri çözülmüş kadar 37 ° C su banyosunda flakon koymak. </li><li> 1. adımı 15ml tüp içine hücreleri dondurma şişe ve yerden çıkarın. </li><li> 180g az 5 dakika süreyle santrifüj, ve sonra süpernatantı atın. </li><li> DMEM + orta 10 ml hücreleri yeniden süspanse edin ve jelatin kaplı 100 mm çanak transfer. Hücrelerin% 80-90 konfluent kadar 37 ° C,% 5 CO 2 inkübatör inkübe edin. </li><li> Geçiş: 293 hücreler PBS ile yıkama hücreleri, PBS aspirat ve 0.05% trypsin/0.53 mM EDTA 10 cm çanak başına 4 ml ve 37 az 1 dakika boyunca inkübe ° C </li><li> 10 ml DMEM + orta tekrar süspansiyon dokunarak yemeklerinden hücreleri, ve 15 ml tüp transfer ayırın. 180g az 5 dakika süreyle santrifüj, süpernatantı aspirat. </li><li> DMEM + orta uygun hacmi ekleyin ve birkaç kez aşağı yukarı pipetleme hücreleri tek bir hücre süspansiyonu break up. </li><li> Hücrelerin sayısını ve FP orta ml başına 8x10 5 hücre konsantrasyonu ayarlayabilirsiniz. </li><li> Tohum hücreleri 100 mm kültür çanak başına 8x10 6 hücreli (10 ml), 37 o gece inkübe ° C,% 5 CO 2. </li></ol> Adım 2: Lentivirüs plazmid ve hasat virüsü ile Transfect 293 hücreleri <ol><li> Her 10 cm plaka için, FUW-Teto-cDNA 10 mikrogram (Oct4, Sox2, Klf4 veya c-Myc) PMD-G 2.5ug ile lentivirüs omurga ve pPax2 7.5 mikrogram kullanın. </li><li> Bir tüp içine üç plazmid aliquoting ederken, DMEM, serum olmadan 500μl içeren ikinci bir tüp içine Fugene 6 transfeksiyon reaktif 30μl teslim ve oda sıcaklığında 5 dakika boyunca parmak-vurma ve inkübe tarafından hafifçe karıştırın. </li><li> DNA karışımı Fugene 6/DMEM-containing tüpe damla bırak, parmak vurma ve 20 dakika inkübe hafifçe karıştırın. </li><li> 293 Kullanıcı çanak içine DNA / Fugene 6 karmaşık damla damla ekleyin ve 37 ° gece inkübe ° C,% 5 CO 2. </li><li> Ertesi sabah: transfeksiyon aspire reaktif içeren orta, 5 ml taze ES hücre orta ekleyin ve hücrelerin inkübatör dönün. </li><li> 48 saat ve 72 saat sonra transfeksiyon, 0.45 mikron gözenek boyutu selüloz asetat filtre üzerinden filtreleme, ve 15 ml tüp aktarılması, 10 ml steril tek kullanımlık şırınga kullanarak 293 orta toplamak. </li><li> Ultrasantrifügasyon tarafından virüs süpernatant Konsantre. Istenilen hacmi virüs pelletini tekrar Oct-3/4-, Sox2, Klf4-c-Myc-lentiviruses içeren orta eşit parçaya amixture. </li><li> Virüs yaparken, (2x10 6 hücre çanak ortalama) 10 cm yemekleri% 90 confluency Oct4-neo/rtTA veya Oct4-GFP/rtTA MEFS (geçit &lt;3) hazırlamak </li><li> Kültür ortamı ve 10 ml PBS ile yıkama aspire. </li><li> 0.05% trypsin/0.53 mM EDTA çanak başına 1 ml ekleyin ve 37 inkübe ° C 10 dakika, PBS atın. </li><li> 9 ml kültür ortamı, hücreler tek bir hücre için askıya alma, ve 15 ml&#39;lik bir tüp transfer. </li><li> Hücrelerin sayısını ve 8x10 ml başına 4 hücreli konsantrasyonunu ayarlamak. Jelatin ile kaplı 10 cm çanak 10 ml (8x10 5 hücre) hücre süspansiyonu aktarın. 37 geceleme çanak inkübe ° C,% 5 CO 2. </li><li> Fibroblast çanak orta aspire ve virüs içeren orta 10 ml ekleyin. 37 gecede 4 saat hücreleri ° C,% 5 CO 2 inkübe edin. </li><li> 24 sonra bir fibroblast çanak orta aspirat ve 10 ml taze ES hücre orta ekleyin. </li><li> Dört genlerin ifade başlatmak için orta içeren Doksisiklin düzenli ES hücre orta yerine, diğer bir deyişle, yeniden programlama başlatmak. </li><li> Oct4-neo muhabiri kullanıyorsanız, daha sonra 6 ve 9 gün arasında zaman ilaç bölümünü başlatın. </li><li> Koloniler aldı yeterince büyük olana kadar her gün orta değiştirin. Kolonileri ilk reprogramming başladıktan sonra yaklaşık bir hafta görünür hale gelir. Bunlar, yaklaşık 20 gün kadar alınır kadar büyük haline gelmelidir. </li></ol> Adım 3: IPS koloniler toplayıp Toplama önce Gün: <ol><li> Γ-ışınlanmış DR4 MEFS ile gerekli sayıda jelatin kaplı 24 kuyucuğu Tohum </li></ol> Gün 1 Klonlar Toplama: <ol><li> 1-2 saat almadan önce plakalar üzerinde koloniler besleyin. </li><li> Bir V-96-iyi dipli çanak kuyulardan 15 ul PBS ekleyin. </li><li> Kolonileri PBS ile bir kez alınır içeren çanak yıkayın ve çanak PBS 10ml eklemek. </li><li> Koloniler küçük 5μl pipetlemeyin ipuçları (4 ul P20 Pipetman ayarlamak) ile seçin. Transferi co96-iyi yemek, iyi bir lonies. </li><li> Tripsin 20 ul ve multi-kanal (12) pipetor kullanarak her ekleyin. Pipetleyin yukarı ve aşağı 3 kez. 37 ° C&#39;de 4 dakika. </li><li> 100 ES orta ul tripsinize koloniler ekleyin. Pipetleyin yukarı ve aşağı 10 kez. 12 yerde pipetor diğer her konumda bir ipucu kullanarak 24-iyi bir yemek için 96-iyi çanak bir defada 6 klonları aktarın. </li><li> 24 plaka hücreler hücrelerin ES hücre orta ile günlük beslenme,% 80-90 confluency ulaşıncaya kadar 37 ° C,% 5 CO2 inkübatör büyütün. Hücreler muhtemelen 3-7 gün içinde genişletmek için hazır olacak. </li></ol> Adım 4 <ol><li> IPS hücreleri genişletilmesi <ol><li> Orta aspire, hücreler ve 1 ml PBS ile yıkayın. </li><li> Tamamen PBS çıkarın, 0.1 ml% 0.25 tripsin / 37 az 1 mM EDTA ve inkübe ° C 10 dakika ekleyin. </li><li> ES orta 0.4 ml ekleyin ve tek hücre süspansiyonu için yukarı ve aşağı pipetleme hücreleri tarafından askıya. </li><li> 6-plaka iyi bir hücre süspansiyonu Transfer, 1.5 ml ES hücre orta ekleyin ve 37 inkübe ° C,% 5 CO2 inkübatör hücrelerin 6-kuyucuğu% 80-90 confluency ulaşana kadar. Bu noktada, aşağıdaki gibi hücreler dondurulmuş stok hazırlamak. </li></ol></li><li> Dondurma stok hazırlanması <ol><li> Orta aspire hücreleri ve 2 ml PBS ile yıkayın. </li><li> Tamamen PBS çıkarın, 0.5 ml% 0.25 tripsin / 37 az 1 mM EDTA ve inkübe ° C 10 dakika ekleyin. </li><li> ES orta 2 ml ekleyin ve tek hücre süspansiyonu için yukarı ve aşağı pipetleme hücreleri tarafından askıya. </li><li> 15 ml&#39;lik bir tüp hücre süspansiyonu Transfer, hücrelerin sayısını ve hücrelerin aşağı döndürün. </li><li> Süpernatantı atın ml başına 2x10 6 hücre konsantrasyonu ES orta hücreleri tekrar süspansiyon haline getirin. </li><li> 2 ES hücre dondurma orta (ES orta% 20 DMSO) ve flakon başına 0.5 ml kısım hazırlayın. </li><li> Şişeleri dondurma ve hafifçe karıştırın 0.5 ml hücre süspansiyonu aktarın. </li><li> Bir hücre dondurma kabı koyun ve şişeleri ° C gecede -80 tutmak, uzun süreli depolama için ertesi gün sıvı azot transfer. </li></ol></li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

Bu video, Oct4-GFP/R26-M2rtTA ve Nanog-GFP/R26-M2rtTA fareler elde edilen MEFS GFP pozitif, pluripotent iPS hücreleri oluşturmak için bir indüklenebilir lentiviral sisteminin nasıl kullanılacağını göstermektedir. Bu prosedür, yeniden programlama işlemi sırasında transgen ekspresyonu kontrolü sağlar çünkü iPS hücreleri üretmek için yararlı bir yöntemdir. IPS hücreleri nesil virüslerin kullanımı bir klinik ortamda bu hücrelerin kullanımını engellemektedir rağmen, bu prosedür henüz tanımlanan yeniden programlama sürecine altını önemli biyolojik sorulardan bazılarını yanıtlamak için etkinleştirin.

<table><tbody><tr><td>Doxycycline hyclate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D9891-100G</td><td></td></tr></tbody></table>

generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

Pluripotency farklılaştırılmış murin Oct4 viral transdüksiyon tarafından indüklenen olabilir, Sox2, Klf4, ve c-Myc (Takahashi ve Yamanaka, 2006; Wernig ve ark, 2007; Okita, vd, 2007;. Maherali, et al, 2007). Biz bu dört transkripsiyon faktörleri doksisiklin ifade edildiği bir yeniden programlama strateji geliştirdiler (dox) lentiviral vektörler (Brambrink ve ark, 2008) indüklenebilir. Bu indüklenebilir yapıları kullanarak, fare embriyonik fibroblastlar (MEFS) alınan uyarılmış pluripotent kök (iPS) hücreleri elde edebilirsiniz. Bu videoda, biz dört transkripsiyon faktörleri ifade ve iPS hücreleri üretmek için bu virüsleri ile enfekte MEFS nasıl göstereceğim indüklenebilir lentiviruses nesil için prosedürü göstermektedir. Indüklenebilir lentiviruses kullanarak, kültür ortamı Doxycyline ek tarafından kontrol dört faktör ifade. Bu sistemin avantajı, geleneksel retroviral enfeksiyon üzerinde, programlama ve gen ekspresyonu gereksinimleri kinetiği ayrıntılı olarak analiz edilebilir kapalı genler açmak ve kapatmak için yeteneğidir.

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Bu video indüklenebilir lentivirüs kullanarak bağlı pluripotent kök hücreleri üretmek için prosedürü ifade Oct4, Sox2, c-Myc ve Klf4 gösterir.

Sox-2 Zorla İfade yoluyla MEFS iPS Hücreler oluşturma, Eki 4, c-Myc ve Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

generating-ips-cells-from-mefs-through-forced-expression-sox-2-oct-4

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

36.2K Görüntüleme.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Bu video indüklenebilir lentivirüs kullanarak bağlı pluripotent kök hücreleri üretmek için prosedürü ifade Oct4, Sox2, c-Myc ve Klf4 gösterir.

Video: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Bütünleşmeyen episomal plasmidler kullanılarak dondurulmuş Buffy tüylerdeki uyarılmış pluripotent kök hücrelerin üretilmesi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Gelişim Biyolojisi

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Bir Transpozon Sistemi Kullanılarak Fare Amniyotik Sıvı Hücreleri pluripotent kök hücrelerin üretimi

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Dört Transkripsiyon Faktör Fare Embriyonik Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi, Doksisiklin lentiviral İletim Sistemi Đndüklenebilir

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Biyoloji

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent İnsan TF lentivirüs İnsan Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi ayarla

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

İnsan iPS hücreleri dondurularak ve kurtarma Tam nakavt Serum Yedek Besleyici-Alerjik Orta

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP Retroviral Vector kullanarak İsteyerek Pluripotent Kök Hücrelerinin (iPSCs) içine İnsan somatik hücreleri yeniden programlama

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Fare Embriyonik Kök Hücre Bakım ve Kendini yenileme Regulate Genler Okumak Oct4GiP Reporter Assay

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCA lentiviral Vektör Kullanma Çevresel Blood İnsan İsteyerek Pluripotent Kök Hücre Üretimi

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Bir programlayabilme Fare Modeli iPS Hücre araürünler Hücre Yüzey Etiket Aracılı Arıtma

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...