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.

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.

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.

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.

Common Recurrent DKC1 Mutation A353V Does Not Support Telomere Maintenance in Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 50-50
Author(s):  
Baiwei Gu ◽  
Jason A. Mills ◽  
Jian-meng Fan ◽  
Deborah L. French ◽  
Monica Bessler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Telomere Length ◽  
Pluripotent Stem Cells ◽  
Skin Fibroblast ◽  
Bone Marrow Failure ◽  
Ips Cells ◽  
Telomere Maintenance ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Abstract Abstract 50 Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome showing considerable genetic and clinical heterogeneity. The most common form is the X-linked form due to mutations in the DKC1 gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Six other genes, all of whose products are involved in telomere maintenance, have been shown to be mutated in DC, together the seven genes accounting for about half of the known cases. The X-linked form can cause severe disease for which therapeutic options are limited. It is known that mutant dyskerin destabilizes telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known. So far studies of the cell biology of DC stem and progenitor cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in telomere biology between mouse and human. An alternative approach that has recently become feasible is the production of induced pluripotent stem cells (iPSC) from patient fibroblasts that can then be used to investigate disease pathogenesis. Accordingly we generated iPSC from skin fibroblast from X-linked DC patients carrying DKC1 mutations Q31E, δ37A and 353V, and by using the classical OCT4, KLF4, SOX2 and cMYC 4-transcription factor system. Of particular interest is the A353V mutation since this is a recurrent mutation and accounts for about 40% of DKC1 mutations. In total, we obtained two Q31E clones, three δ37 clones and eight A353V clones. We found that all these DKC1 mutant iPS cells express decreased levels of dyskerin, in agreement with our mouse studies that show mutant proteins are relatively unstable. Mutant iPSC have very low levels of TERC (only 20–30% of the levels in WT iPSC) while TERT expression is the same as in WT cells. By using the TRAP assay, we found that both A353V and δ37 iPSC showed dramatically decreased telomerase activity; only 10–20 % compared to WT iPSC. After measuring the telomere length of both patient skin fibroblast cells and DKC1 mutant iPSC, we found A353V and δ37 iPSC lost the ability to elongate the telomere end during iPSC reprogramming while WT iPSC showed significantly increased telomere length compared to WT skin fibroblast cells. These results indicated that DKC1 iPSC are defective in telomere maintenance. In terms of ribosome biogenesis, we found that some snoRNA expression was slightly decreased including H/ACA snoRNAs E2, E3, U69, ACA10 and scaRNAs U90 and U93 while all C/D snoRNA we investigated were unchanged compared with WT iPS cells. We also found that DKC1 mutant iPS cells did not show significantly changes in ribosomal profiles or in the kinetics of rRNA processing. Together these results suggest that the iPSC faithfully reproduce the molecular features of the human disease and will prove to be a useful tool in investigations of the pathogenesis and treatment of DC. Disclosures: Bessler: Alexion Phamaceutical: Membership on an entity's Board of Directors or advisory committees; National Organization for Rare Dieases: Speakers Bureau.

scholarly journals Potential and Limitation of HLA-Based Banking of Human Pluripotent Stem Cells for Cell Therapy

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Casimir de Rham ◽  
Jean Villard
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Human Leukocyte ◽  
Ips Cells ◽  
Abo Blood Group ◽  
Leukocyte Antigen ◽  
Induced Pluripotent ◽  
The One ◽  
Standard Medication

Great hopes have been placed on human pluripotent stem (hPS) cells for therapy. Tissues or organs derived from hPS cells could be the best solution to cure many different human diseases, especially those who do not respond to standard medication or drugs, such as neurodegenerative diseases, heart failure, or diabetes. The origin of hPS is critical and the idea of creating a bank of well-characterized hPS cells has emerged, like the one that already exists for cord blood. However, the main obstacle in transplantation is the rejection of tissues or organ by the receiver, due to the three main immunological barriers: the human leukocyte antigen (HLA), the ABO blood group, and minor antigens. The problem could be circumvented by using autologous stem cells, like induced pluripotent stem (iPS) cells, derived directly from the patient. But iPS cells have limitations, especially regarding the disease of the recipient and possible difficulties to handle or prepare autologous iPS cells. Finally, reaching standards of good clinical or manufacturing practices could be challenging. That is why well-characterized and universal hPS cells could be a better solution. In this review, we will discuss the interest and the feasibility to establish hPS cells bank, as well as some economics and ethical issues.

scholarly journals Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

2012 ◽  
Vol 14 (4) ◽  
pp. 574-579 ◽  
Author(s):  
Yong Zhu ◽  
Hong-Liang Hu ◽  
Peng Li ◽  
Shi Yang ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
In Vivo Study ◽  
Male Germ Cells ◽  
Induced Pluripotent
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