1.Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro.
Li Jun FANG ; Zi Bei FENG ; Jing Yi MEI ; Jia Hui ZHOU ; Zhan Yi LIN
Journal of Southern Medical University 2022;42(6):929-936
OBJECTIVE:
To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.
METHODS:
EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.
RESULTS:
The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).
CONCLUSION
Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
Echinomycin/metabolism*
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Embryoid Bodies/metabolism*
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Humans
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Hypoxia/metabolism*
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Induced Pluripotent Stem Cells/metabolism*
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beta Catenin/metabolism*
2.The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting.
Yuhan CHEN ; Jiansen LU ; Yanwen XU ; Yaping HUANG ; Dazhuang WANG ; Peiling LIANG ; Shaofang REN ; Xuesong HU ; Yewen QIN ; Wei KE ; Ralf JAUCH ; Andrew Paul HUTCHINS ; Mei WANG ; Fuchou TANG ; Xiao-Yang ZHAO
Protein & Cell 2023;14(7):477-496
Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.
Male
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Mice
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Animals
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Cellular Reprogramming/genetics*
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Tetraploidy
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Pluripotent Stem Cells/metabolism*
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Induced Pluripotent Stem Cells/metabolism*
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DNA Methylation
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Spermatogonia/metabolism*
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Germ Cells/metabolism*
3.Establishment of an internal control for directed differentiation using pluripotent stem cell lines derived from heterozygotic twins.
Yu-mei LUO ; Yong FAN ; Xin-jie CHEN ; Lei YUE ; Qing LI ; Wen-zhi HE ; Xiao-yan MA ; Yu-hong ZHENG ; Xiao-fang SUN
Chinese Journal of Medical Genetics 2012;29(4):398-403
OBJECTIVETo reprogram amniotic fluid cells into pluripotent stem cells in order to create an optimal internal control model for directed cell differentiation.
METHODSHuman amniotic fluid-derived cells (hAFDCs) from heterozygotic twin fetuses were induced by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. In vivo pluripotency, differentiation capacity and karyotype of hAFDCs induced pluripotent stem cells (hAFDCs-iPSCs) were determined.
RESULTShAFDC-iPSCs derived from heterozygotic twins have maintained self renewal, with expression of high pluripotency marker gene detected at both mRNA and protein levels. The cells have maintained their differentiation capacity both in vitro and vivo, and showed normal karyotypes after long-term culturing in vitro.
CONCLUSIONhAFDCs-iPSCs derived from heterozygotic twins have good consistency in terms of genetic background, and can provide a good internal control for directed differentiation of iPSCs, and may be used an ideal source for autologous cell replacement therapy in the later life of the fetus.
Amniotic Fluid ; cytology ; metabolism ; Cell Differentiation ; genetics ; Cell Line ; Female ; Fetus ; metabolism ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Karyotype ; Pluripotent Stem Cells ; cytology ; metabolism ; Pregnancy ; Twins
4.Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy.
Yang YANG ; Yang LIU ; Min CHEN ; Shuangpeng LI ; Xuan LU ; Yu HE ; Kun ZHANG ; Qingjian ZOU
Chinese Journal of Biotechnology 2023;39(10):4098-4107
Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Humans
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Induced Pluripotent Stem Cells
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Sirolimus/metabolism*
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Caspase 9/metabolism*
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RNA, Guide, CRISPR-Cas Systems
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Pluripotent Stem Cells/metabolism*
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Cell Differentiation
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Puromycin/metabolism*
5.Stem Cell Properties of Therapeutic Potential.
The Korean Journal of Gastroenterology 2011;58(3):125-132
Stem cell research is a innovative technology that focuses on using undifferentiated cells able to self-renew through the asymmetrical or symmetrical divisions. Three types of stem cells have been studied in laboratory including embryonic stem cell, adult stem cells and induced pluripotent stem cells. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass and it can give rise to any fetal or adult cell type. Adult stem cells are multipotent, have the ability to differentiate into a limited number of specialized cell types, and have been obtained from the bone marrow, umbilical cord blood, placenta and adipose tissue. Stem cell therapy is the most promising therapy for several degenerative and devastating diseases including digestive tract disease such as liver failure, inflammatory bowel disease, Celiac sprue, and pancreatitis. Further understanding of biological properties of stem cells will lead to safe and successful stem cell therapies.
Adult Stem Cells/cytology/metabolism/transplantation
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Embryonic Stem Cells/cytology/metabolism/transplantation
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Humans
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Induced Pluripotent Stem Cells/cytology/metabolism/transplantation
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Stem Cells/*cytology/metabolism
6.Evolution of iPSC disease models.
Weiqi ZHANG ; Zhichao DING ; Guang-Hui LIU
Protein & Cell 2012;3(1):1-4
7.Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury.
Protein & Cell 2017;8(4):273-283
Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.
Animals
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Cellular Reprogramming
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drug effects
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Cellular Reprogramming Techniques
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methods
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Induced Pluripotent Stem Cells
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cytology
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metabolism
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Mice
8.Three dimensional collagen scaffolds promote iPSC induction with higher pluripotency.
Qi GU ; He ZHU ; Lei CHEN ; Ling SHUAI ; Jinhui FANG ; Jun WU ; Lei LIU ; Wei LI ; Jianwu DAI ; Jie HAO ; Qi ZHOU
Protein & Cell 2016;7(11):844-848
10.Reevaluation of the safety of induced pluripotent stem cells: a call from somatic mosaicism.
Wensu LIU ; Ming LI ; Jing QU ; Fei YI ; Guang-Hui LIU
Protein & Cell 2013;4(2):83-85
Recent studies have been raising doubts on the safety of induced pluripotent stem cells (iPSCs) and proposing that the process of reprogramming brought about copy number variations (CNVs) in iPSCs. However, a recent paper published in Nature provided evidence showing that most CNVs were pre-existed as somatic mosaicism but not resulted from the reprogramming. This new finding would profoundly reshape some previous thoughts and endorse the confidence of iPSCs in both research and therapy.
Cellular Reprogramming
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DNA Copy Number Variations
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Humans
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Induced Pluripotent Stem Cells
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metabolism
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Mosaicism
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Regenerative Medicine