scholarly journals Induced pluripotent stem cells: opportunities and challenges

2011 ◽  
Vol 366 (1575) ◽  
pp. 2198-2207 ◽  
Author(s):  
Keisuke Okita ◽  
Shinya Yamanaka
Keyword(s):  
Stem Cells ◽  
Transient Expression ◽  
Pluripotent Stem Cells ◽  
Current Knowledge ◽  
Epigenetic Modification ◽  
Somatic Cells ◽  
Ips Cells ◽  
Genomic Alteration ◽  
Reprogramming Factors ◽  
Induced Pluripotent

Somatic cells have been reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors, such as Oct3/4 , Sox2 , Klf4 and c-Myc . Induced pluripotent stem (iPS) cells from a patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into the host genomes and may increase the risk of tumour formation. Several non-integration methods have been reported to overcome the safety concern associated with the generation of iPS cells, such as transient expression of the reprogramming factors using adenovirus vectors or plasmids, and direct delivery of reprogramming proteins. Although these transient expression methods could avoid genomic alteration of iPS cells, they are inefficient. Several studies of gene expression, epigenetic modification and differentiation revealed the insufficient reprogramming of iPS cells, thus suggesting the need for improvement of the reprogramming procedure not only in quantity but also in quality. This report will summarize the current knowledge of iPS generation and discuss future reprogramming methods for medical application.

181 Establishment of Induced Pluripotent Stem Cells from Fishing Cat and Clouded Leopard Using Integration-Free Method for Wildlife Conservation

2018 ◽  
Vol 30 (1) ◽  
pp. 230 ◽  
Author(s):  
W. Sukparangsi ◽  
R. Bootsri ◽  
W. Sikeao ◽  
S. Karoon ◽  
A. Thongphakdee
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Dermal Fibroblast ◽  
Ips Cells ◽  
Ips Cell ◽  
Clouded Leopard ◽  
Thermo Fisher Scientific ◽  
Reprogramming Factors ◽  
Induced Pluripotent ◽  
Fisher Scientific

Fishing cat (Prionailurus viverrinus) and clouded leopard (Neofelis nebulosa) are wild felids, currently in vulnerable status according to the International Union for Conservation of Nature red list (2017). Several measures in assisted reproductive technology (e.g. AI, embryo transfer) have been used by the Zoological Park Organization of Thailand (ZPO) to increase their offspring in captivity. Recently, the generation of induced pluripotent stem cell (iPS cells) becomes popular and provides alternative way to preserve good genetics in the form of cell with diverse capacities. This great potential of iPS cells is unlimited self-renewal and pluripotency, similar to embryonic stem cells (ESC). Under the right cell culture conditions, pluripotent stem cells can differentiate into all cell types of the body. Here, we aimed to find the optimal condition to generate integration-free iPS cells from fishing cat and clouded leopard. At first, to obtain somatic cells for cellular reprogramming, adult dermal fibroblast cell lines from both species were established from belly skin tissues. Subsequently, several nucleofection programs of AmaxaTM 4D-nucleofectorTM (Lonza, Basel, Switzerland) were examined to introduce integration-free DNA vectors carrying reprogramming factors into the felid fibroblasts. The transfected cells were cultured under numerous conditions: (1) matrix/defined surface including irradiated mouse embryonic fibroblast, gelatin, vitronectin, and Geltrex® (Thermo Fisher Scientific, Waltham, MA, USA); (2) ESC/iPS cell medium including Essential 8TM (Thermo Fisher Scientific) DMEM containing KnockOutTM Serum Replacement (KOSR; Thermo Fisher Scientific) and/or fetal bovine serum (FBS); and (3) supplement including basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), l-ascorbic acid, nicotinamide, ALK5 inhibitor (A83-01) and RevitaCellTM (Thermo Fisher Scientific). We found that optimal nucleofection programs for human dermal fibroblast including FF-135 and EN-150 were able to transfer episomal vectors and excisable piggyBAC transposon carrying reprogramming factors into fishing cat and clouded leopard fibroblasts, respectively. The iPS-like colonies appeared around 26 to 30 days post-nucleofection. The culture of transfected cells on either Geltrex® or Vitronectin-coated surface supports the formation of iPS-like colonies with different derivation efficiency (0.01 and 0.005%, respectively). In addition, all colonies were formed under medium containing FBS, together with both bFGF and LIF supplements. Taken together, we have developed a platform to generate iPS cells from tissue collection to the establishment of iPS cell culture. This will further enable us to apply the technique to obtain iPS cells from other endangered and vulnerable felid species.

New windows to enhance direct reprogramming of somatic cells towards induced pluripotent stem cells

2012 ◽  
Vol 90 (2) ◽  
pp. 115-123 ◽  
Author(s):  
Saeideh Nakhaei-Rad ◽  
Ahmad R. Bahrami ◽  
Mahdi Mirahmadi ◽  
Maryam M. Matin
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Tumor Development ◽  
Direct Reprogramming ◽  
Slow Kinetics ◽  
Reprogramming Factors ◽  
Low Efficiency ◽  
Induced Pluripotent

Induced pluripotent stem cells are generated by direct reprogramming of somatic cells with the introduction of defined transcription factors or other means. Clinical applications of induced pluripotent stem cells are the latest of stem cell therapy approaches due to overcoming problems associated with insufficient cells from conventional sources and immune rejections. In practice, this is restricted by 4 major barriers including the use of genetic manipulations for delivering the reprogramming factors, low efficiency of this process, slow kinetics of the direct reprogramming, and potential for tumor development. Here, we review the latest achievements in improving reprogramming efficiency by alternative strategies. These alternatives mainly involve the replacement of genetic reprogramming factors with small molecules or other factors.

388 OPTIMIZATION OF INDUCED PLURIPOTENT STEM CELL GENERATION FROM BOVINE FIBROBLASTS

2010 ◽  
Vol 22 (1) ◽  
pp. 350
Author(s):  
M. L. Lim ◽  
H. Sumer ◽  
J. Liu ◽  
P. J. Verma
Keyword(s):  
Stem Cells ◽  
Cell Density ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
Alternative Methods ◽  
Packaging Cell Line ◽  
A Cell ◽  
Reprogramming Factors ◽  
Induced Pluripotent ◽  
Adult Fibroblasts

Difficulties associated with isolation and culturing bovine embryonic stem (ES) cells has led to the exploration of alternative methods for generating pluripotent stem cells. The viral delivery of reprogramming factors Oct4, Sox2, cMyc, and Klf4 has resulted in generation of induced pluripotent stem cells (iPS) in rodent, human, rhesus monkey, and pig somatic cells. In the current study, we improved the efficiency of retroviral transduction of bovine adult fibroblasts (BAF) for the generation of bovine iPS cells. Bovine adult fibroblasts were transduced with 4 human factors: Oct 4, Sox 2, cMyc, and Klf4. To determine transfection efficiency, pMXs-GFP was used as a reporter. The effect of change in cell density of the Platinum A retroviral packaging cell line (Plat A), cell densities of the target BAF, and infection regimes on the transfection rates was examined. A reduction in Plat A cell density from 8 × 106 to 2 × 106 did not alter transfection rates. Reduced target cell density from 4 × 105 to 4 × 104 (10-fold) improved the transfection rates from 0.31 to 7.06%, P < 0.001 (n = 3). Subjecting the BAF to 2 sequential rounds of viral transduction further improved the transfection rates to 13.88%, P < 0.001 (n = 3). These preliminary results suggest that optimizing the density of target cells can greatly improve transduction outcomes. Following viral induction with the 4 reprogramming factors, putative bovine iPS colonies were observed when the transfection rate was >1%. The putative bovine iPS cells were cultured in alpha-minimal essential medium supplemented with 20% FCS and 10 ng mL-1 human leukemia inhibitory factor. These putative bovine iPS colonies had mouse ES-like morphology, were multilayered, and had high nucleus-to-cytoplasmic ratio. They stained positive for alkaline phosphatase activity. The colonies were manually passaged onto mitomycin C-inactivated mouse embryonic fibroblasts every 5 to 7 days but could only be expanded for a limited number of passages. Other strategies are currently being explored to improve stable reprogramming of BAF such as epigenetic modification of cells, lentivirus-mediated transduction, and investigation of media suitable to maintain putative bovine iPS colonies for further characterization including RT-PCR or immunohistochemical detection for pluripotent markers and in vivo differentiation ability. Acknowledgments are given to Dairy Australia.

scholarly journals MicroRNAs and Induced Pluripotent Stem Cells for Human Disease Mouse Modeling

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Chingiz Underbayev ◽  
Siddha Kasar ◽  
Yao Yuan ◽  
Elizabeth Raveche
Keyword(s):  
Stem Cells ◽  
Mouse Models ◽  
Pluripotent Stem Cells ◽  
Human Disease ◽  
Disease Modeling ◽  
Somatic Cells ◽  
Ips Cells ◽  
Potential Candidate ◽  
Genetically Engineered Mice ◽  
Induced Pluripotent

Human disease animal models are absolutely invaluable tools for our understanding of mechanisms involved in both physiological and pathological processes. By studying various genetic abnormalities in these organisms we can get a better insight into potential candidate genes responsible for human disease development. To this point a mouse represents one of the most used and convenient species for human disease modeling. Hundreds if not thousands of inbred, congenic, and transgenic mouse models have been created and are now extensively utilized in the research labs worldwide. Importantly, pluripotent stem cells play a significant role in developing new genetically engineered mice with the desired human disease-like phenotype. Induced pluripotent stem (iPS) cells which represent reprogramming of somatic cells into pluripotent stem cells represent a significant advancement in research armament. The novel application of microRNA manipulation both in the generation of iPS cells and subsequent lineage-directed differentiation is discussed. Potential applications of induced pluripotent stem cell—a relatively new type of pluripotent stem cells—for human disease modeling by employing human iPS cells derived from normal and diseased somatic cells and iPS cells derived from mouse models of human disease may lead to uncovering of disease mechanisms and novel therapies.

scholarly journals Biological importance of OCT transcription factors in reprogramming and development

2021 ◽  
Author(s):  
Kee-Pyo Kim ◽  
Dong Wook Han ◽  
Johnny Kim ◽  
Hans R. Schöler
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Donor Cell ◽  
Structural Components ◽  
Oct4 Expression ◽  
Reprogramming Factors ◽  
Induced Pluripotent

AbstractEctopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.

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