Induced Pluripotent Stem Cells(iPS Cells):Current Status and Future Prospect*

Abstract. Induced pluripotent stem cells (iPS cells) generated by cellular reprogramming from nonhuman primates (NHPs) are of great significance for regenerative medicine and for comparative biology. Autologously derived stem cells would theoretically avoid any risk of rejection due to host–donor mismatch and may bypass the need for immune suppression post-transplant. In order for these possibilities to be realized, reprogramming methodologies that were initially developed mainly for human cells must be translated to NHPs. NHP studies have typically used pluripotent cells generated from young animals and thus risk overlooking complications that may arise from generating iPS cells from donors of other ages. When reprogramming is extended to a wide range of NHP species, available donors may be middle- or old-aged. Here we have pursued these questions by generating iPS cells from donors across the life span of the common marmoset (Callithrix jacchus) and then subjecting them to a directed neural differentiation protocol. The differentiation potential of different clonal cell lines was assessed using the quantitative polymerase chain reaction. The results show that cells derived from older donors often showed less neural marker induction. These deficits were rescued by a 24 h pretreatment of the cells with 0.5 % dimethyl sulfoxide. Another NHP that plays a key role in biological research is the chimpanzee (Pan troglodytes). iPS cells generated from the chimpanzee can be of great interest in comparative in vitro studies. We investigated if similar deficits in differentiation potential might arise in chimpanzee iPS cells reprogrammed using various technologies. The results show that, while some deficits were observed in iPS cell clones generated using three different technologies, there was no clear association with the vector used. These deficits in differentiation were also prevented by a 24 h pretreatment with 0.5 % dimethyl sulfoxide.

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Non-immunogenic Induced Pluripotent Stem Cells, a Promising Way Forward for Allogenic Transplantations for Neurological Disorders

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.623717 ◽

2021 ◽

Vol 2 ◽

Author(s):

Henriette Reventlow Frederiksen ◽

Ulrik Doehn ◽

Pernille Tveden-Nyborg ◽

Kristine K. Freude

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Neurological Disorders ◽

Developmental Disorders ◽

Gene Editing ◽

Current Status ◽

Cell Replacement ◽

Induced Pluripotent ◽

The Brain

Neurological disorder is a general term used for diseases affecting the function of the brain and nervous system. Those include a broad range of diseases from developmental disorders (e.g., Autism) over injury related disorders (e.g., stroke and brain tumors) to age related neurodegeneration (e.g., Alzheimer's disease), affecting up to 1 billion people worldwide. For most of those disorders, no curative treatment exists leaving symptomatic treatment as the primary mean of alleviation. Human induced pluripotent stem cells (hiPSC) in combination with animal models have been instrumental to foster our understanding of underlying disease mechanisms in the brain. Of specific interest are patient derived hiPSC which allow for targeted gene editing in the cases of known mutations. Such personalized treatment would include (1) acquisition of primary cells from the patient, (2) reprogramming of those into hiPSC via non-integrative methods, (3) corrective intervention via CRISPR-Cas9 gene editing of mutations, (4) quality control to ensure successful correction and absence of off-target effects, and (5) subsequent transplantation of hiPSC or pre-differentiated precursor cells for cell replacement therapies. This would be the ideal scenario but it is time consuming and expensive. Therefore, it would be of great benefit if transplanted hiPSC could be modulated to become invisible to the recipient's immune system, avoiding graft rejection and allowing for allogenic transplantations. This review will focus on the current status of gene editing to generate non-immunogenic hiPSC and how these cells can be used to treat neurological disorders by using cell replacement therapy. By providing an overview of current limitations and challenges in stem cell replacement therapies and the treatment of neurological disorders, this review outlines how gene editing and non-immunogenic hiPSC can contribute and pave the road for new therapeutic advances. Finally, the combination of using non-immunogenic hiPSC and in vivo animal modeling will highlight the importance of models with translational value for safety efficacy testing; before embarking on human trials.

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GENERATION AND CHARACTERISATION OF INDUCED PLURIPOTENT STEM CELLS (iPSCs) FROM ADULT CANINE FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab244 ◽

2012 ◽

Vol 24 (1) ◽

pp. 285

Author(s):

Jorge A. Piedrahita ◽

Sehwon Koh ◽

Natasha Olby

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Es Cells ◽

Ips Cells ◽

Embryoid Bodies ◽

Germ Layers ◽

Induced Pluripotent ◽

Derivatives Of

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). In his presentation we will describe the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured in FGF2 plus LIF showed strong AP activity expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28 as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). In vitro differentiation by formation of embryoid bodies (EBs) and directed differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 (endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic protein (GFAP), TUJ1, NCAM and bIII-tubulin (TUBB, ectoderm). In vivo, the putative canine iPS cells formed simple teratomas that expressed markers for all three germ layers. In summary, we were able to derive induced pluripotent cells from adult somatic cells by using four transcription factors. The isolated canine iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF and LIF is still unknown. This work was funded by a grant from the America Kennel Club to JAP.

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186 Improving Success Rate of Establishment and Maintenance of Porcine Induced Pluripotent Stem Cells by Investigation of Colony Morphology

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab186 ◽

2018 ◽

Vol 30 (1) ◽

pp. 233

Author(s):

P. Setthawong ◽

P. Phakdeedindan ◽

M. Techakumphu ◽

T. Tharasanit

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Induced Pluripotent Stem Cells ◽

Success Rate ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Ips Cells ◽

Fibroblast Cells ◽

Induced Pluripotent

Induced pluripotent stem cells (iPS cells) are generated by reprogramming of somatic cells using ectopic introduction of 4 transcription factors, including OCT4, SOX2, KLF4, and c-MYC (OSKM). Fibroblast cells are the most commonly used as a primary cell source for iPS cells because they are easy to harvest and culture. However, reprogramming efficiency of porcine fibroblasts is poor (~0.1%). During reprogramming process, mixed populations of primary colonies become the major obstacle in iPS establishment. In this study, we characterised 2 different colony morphologies at colony pick-up (compact and loose iPS morphology). We hypothesised that compact colonies will proceed to long-term culture and pluripotency. The fibroblast cells were isolated from tails of piglets and transfected with retroviral vectors expressing OSKM. The primary colonies were counted on Day 7 after gene transduction. The iPS-like colonies were divided into compact (n = 10) and loose (n = 10) morphology at colony pick-up. The characteristics of iPS-like cell lines were analysed by morphology, alkaline phosphatase staining, G-banding karyotype, immunofluorescence staining (OCT4), pluripotent gene expression (RT-PCR), and embryoid body formation. A total of 1,697 iPS-like colonies (2.34%) were observed. The compact colonies contained with tightly packed cells with distinct border between iPS colony and feeder cells, while colonies with irregular shape and border were classified as loose colonies. These 2 types of iPS-like colonies expressed alkaline phosphatase and OCT4. A total 5 of 10 (50%) compact morphology cell lines could be maintained in the undifferentiated state for more than 50 passages. But only 3 of 10 (30%) loose morphology cell lines demonstrated pluripotent characteristics. G-Banding karyotype analysis revealed normal chromosome number (n = 38). All of the cell lines also expressed endogenous pluripotent genes, including OSKM and NANOG and formed three-dimensional aggregating masses. In this study, we found that the cell lines from compact morphology could be maintained for longer than those of the loose morphology. A high rate of differentiation of loose iPS colony may also indicate that this type of colony has different pluripotency signals or incomplete reprogramming compared with compact colony. In conclusion, selection of compact colony morphology at colony pick-up is simple and reflects long-term propagation of porcine iPS cell lines. This information is important for improving the success rate of establishment and maintenance of porcine iPS cells.

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Application of Cord Blood and Cord Blood-Derived Induced Pluripotent Stem Cells for Cartilage Regeneration

Cell Transplantation ◽

10.1177/0963689718794864 ◽

2018 ◽

Vol 28 (5) ◽

pp. 529-537 ◽

Cited By ~ 3

Author(s):

Yeri Alice Rim ◽

Yoojun Nam ◽

Ji Hyeon Ju

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Articular Cartilage ◽

Cord Blood ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

Current Status ◽

Induced Pluripotent

Regeneration of articular cartilage is of great interest in cartilage tissue engineering since articular cartilage has a low regenerative capacity. Due to the difficulty in obtaining healthy cartilage for transplantation, there is a need to develop an alternative and effective regeneration therapy to treat degenerative or damaged joint diseases. Stem cells including various adult stem cells and pluripotent stem cells are now actively used in tissue engineering. Here, we provide an overview of the current status of cord blood cells and induced pluripotent stem cells derived from these cells in cartilage regeneration. The abilities of these cells to undergo chondrogenic differentiation are also described. Finally, the technical challenges of articular cartilage regeneration and future directions are discussed.

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