scholarly journals Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus

2018 ◽  
Vol 50 (4) ◽  
pp. 1318-1331 ◽  
Author(s):  
Huahu Ye ◽  
Qiwei Wang
Keyword(s):  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Clinical Applications ◽  
Cell Reprogramming ◽  
Peripheral Blood Cells ◽  
Cell Recovery ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Immunocompromised Mice

Background/Aims: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. Methods: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. Results: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. Conclusion: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications.

scholarly journals Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells

2010 ◽  
Vol 7 (1) ◽  
pp. 20-24 ◽  
Author(s):  
Judith Staerk ◽  
Meelad M. Dawlaty ◽  
Qing Gao ◽  
Dorothea Maetzel ◽  
Jacob Hanna ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Cells ◽  
Induced Pluripotent

An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells

Stem Cells ◽  
2013 ◽  
Vol 31 (3) ◽  
pp. 458-466 ◽  
Author(s):  
Keisuke Okita ◽  
Tatsuya Yamakawa ◽  
Yasuko Matsumura ◽  
Yoshiko Sato ◽  
Naoki Amano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Peripheral Blood Cells ◽  
Induced Pluripotent

scholarly journals Cell reprogramming shapes the mitochondrial DNA landscape

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Wei ◽  
Daniel J. Gaffney ◽  
Patrick F. Chinnery
Keyword(s):  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Cell Reprogramming ◽  
Protein Coding ◽  
Mtdna Mutations ◽  
Age Related ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Mtdna Variants ◽  
Original Donor

AbstractIndividual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10−5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.

scholarly journals Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells

2019 ◽  
Vol 20 (4) ◽  
pp. 810 ◽  
Author(s):  
Chizuru Iwasawa ◽  
Ryota Tamura ◽  
Yuki Sugiura ◽  
Sadafumi Suzuki ◽  
Naoko Kuzumaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Clinical Applications ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Simplex Virus

Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a “thymidine block”, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.

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