Development and characterization of induced pluripotent stem cells ipscs derived from human oral squamous carcinoma cell oscc nalini devi verusingam induced pluripotent stem cells ipscs technology pioneered by yamanaka and group had offered a new insight to study the pathophysiology of cancer. Dopaminergic neuron differentiation of human pluripotent stem cells. The availability of robust and short protocols to induce monocytes and macrophages from human induced pluripotent stem cells hipscs will benefit many applications of immune cells in biomedical research. A bioinformatics assay for pluripotency in human cells. Hence, a new in vitro model comprising the advantages of 3d cell culture and the availability of induced pluripotent stem cells ipscs of human origin was developed and characterized. Antibodybased tools and protocols for characterization of pluripotent stem cells. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patientspecific induced pluripotent stem cells ipscs has become possible in multiple species. Schwartz, c timothy hacker, d vernella vickerman, a scott swanson, a ning leng, a, e bao kim nguyen, a angela elwell, a jennifer bolin, a matthew e. Functional characterization of human pluripotent stem cell. Phenotypic characterization of toxic compound effects on. Characterization of monocytes and macrophages derived from human induced pluripotent stem cells xucao,1 francijnae. Pluripotent stem cell product guide thermo fisher scientific. Chapter characterization and criteria of embryonic.
Keywords induced pluripotent stem cells, reprogramming, reprogramming factors, embryonic stem cells, gene editing technology, crispr, cell therapy howtocite this article omole and fakoya2018, tenyearsof progress and promise of induced pluripotent stem cells. Isolation, culture, and functional characterization of. Functional characterization of induced pluripotent stem cell. Previous work has shown that adult somatic human dermal fibroblasts can be efficiently reprogrammed into ipscs through exogenous expression of four transcription factors oct4, klf4, cmyc, and sox2 with a single polycistronic lentivirus, or stemcca, flanked by loxp sites hstemccaloxp. A facs analysis of monocytes markers cd14, cd45, and integrins cd49d, cd18, cd29, and cd11b on freshly isolated and. Functional characterization of human pluripotent stem. The cells must be shown to be safe for clinical use and not produce tumors or differentiate into unwanted cell types. These stem cells are similar to embryonic stem es cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Pluripotent stem cell characterization methods youtube. It has been shown that the epigenetic status, such as the presence of dna methylation and histone modifications, changes dramatically during ips cell generation. Generation and functional characterization of monocytes and. Reference maps of human es and ips cell variation enable highthroughput characterization of stem cells. Methods and protocols highlights the best methods and systems for the entire work flow. Breton a, sharma r, diaz ac, parham ag, graham a, neil c, et al.
The resulting induced pluripotent stem cells ipscs show many of the characteristics of escs, including similar marker expression, the ability to differentiate into all three germ layers and, in the case of mouse cells, to generate germline chimeras. Human cms derived from ipscs were studied in standard 2d culture and as cardiac microtissues mts formed in hanging drops. Takahashi k, yamanaka s 2006 induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. The journey of hesc research is not that smooth, as it has faced several challenges which are limited to not only tumor. Electrophysiologic characterization of calcium handling in. Since the first generation of embryonic stem cells escs1,2 and induced pluripotent stem cells ipscs3,4, numerous cell culture techniques focusing on the culture of these cells have been pre sented in detail5,6. Derivation and characterization of induced pluripotent stem cells from equine fibroblasts. The journey of hesc research is not that smooth, as it has faced several challenges which are limited to not only tumor formation and immunorejection. Molecular analysis tools for epigenomic characterization. Induced pluripotent stem cells ipscs are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patientspecific manner. Characterization and comparison of osteoblasts derived from. Human pluripotent stem cells hpsc, which include human embryonic stem cells hesc and human induced pluripotent stem cells hipsc, have the ability to indefinitely proliferate and differentiate to derivatives of all three germ layers. However, the barriers along the translational pipeline are still numerous. New developments in stem cell biology offer alternatives for the reconstruction of criticalsized bone defects.
Characterization and therapeutic application of mesenchymal. Human embryonic stem cells hescs hold great potential for the treatment of various degenerative diseases. Characterization and therapeutic application of mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells huiyan wang 1, dairui li 1, zhichen zhai 1, xiaoran zhang 1, weijun huang 1, xiaoyong chen 1, lihua huang 2, huanyao liu 1, jiaqi sun 1, zhengwei zou 1, yubao fan 1, qiong ke 1,3, xingqiang lai 1. Several reports have documented the derivation of pluripotent cells multipotent germline stem cells from spermatogonial stem cells obtained from the adult mouse testis. Since the first generation of embryonic stem cells escs1,2 and induced pluripotent stem cells ipscs 3,4, numerous cell culture techniques focusing on the culture of these cells have been pre. Microrna characterization in equine induced pluripotent. Ten years of progress and promise of induced pluripotent. Aneuploidy in pluripotent stem cells and implications for. Generation and functional characterization of monocytes. Highthroughput and costeffective characterization of.
Development and characterization of a scaffoldfree 3d. Isolation and characterization of pluripotent human. While it is believed that a significant percentage of cardiovascular diseases are due to environmental. Freitas, sarah burton, christie munn, michael hancock, kwi. The characteristics of pluripotent stem cells which make them so ideal for clinical use e. Human stem cellderived tissue cells may provide such a model. Highthroughput and costeffective characterization of induced pluripotent stem cells author links open overlay panel matteo dantonio 1 grace woodruff 2 jason l. Electrophysiologic characterization of calcium handling in human induced pluripotent stem cellderived atrial cardiomyocytes previous article unraveling the inconsistencies of cardiac differentiation efficiency induced by the gsk3. Contents introduction 4 1 reprogramming of human fibroblasts 7 2 selection of ipsc clones for feederdependent culture 21 3 preparation of feeder layer 25 4 feederdependent culture of human induced pluripotent stem cells hipscs 29 5 transition of mature ipscs to feederfree culture system 39 6 culture, expansion, and cryopreservation of pluripotent stem cells in feeder. They may therefore represent a potentially ideal source of homogeneous and highquality msc. Functional characterization of induced pluripotent stem.
Generation and characterization of transgenefree human. Pradhan joined thermo fisher scientific in 2016 and is currently working on next generation tools and technologies development with the goal of providing assays and methods for molecular characterization of stem cells and t cells utilizing methylation platform, arraybased whole genome and transcriptomic profiling and taqman qpcr assays. These cells had the capability to differentiate into derivatives of all three germ layers endoderm, ectoderm, and mesoderm, self. In this manuscript, we describe the detailed characterization of the two ipsc clones generated using this process, including whole.
Jul 01, 2005 this is similar to the reported characterization of undifferentiated primate es and parthenogenetic stem cells 7, human es cells, and ntes cells 9, 11, 20 but is different from the previously established pluripotent stem cell lines produced from bovine ivf embryos, which were ssea1 positive 14, 17. To this end, we differentiated baboon fibroblastderived induced pluripotent stem cells bipscs into dopaminergic neurons with the application of specific morphogens and growth factors. Generation and characterization of pluripotent stem cells. Divided into four convenient sections, topics include a focus on producing ipsc from diverse somatic sources, media systems for expanding esc and ipsc with detailed protocols for directed differentiation into specific lineages. One of these developments is the use of induced pluripotent stem ips cells. Since the first generation of embryonic stem cells escs1,2 and induced pluripotent stem cells ipscs 3,4, numerous cell culture techniques focusing on. Pdf characterization of cancer stemlike cells derived from. However, a current limitation to using induced pluripotent stem cells ipscs to model human disease is the timeinefficiency and cost of standard characterization methods required after reprogramming. Generation and characterization of induced pluripotent. Monocytes and macrophages are essential for immune defense and tissue hemostasis. They are also the underlying trigger of many diseases. Here, we combine singlecell rna sequencing and crisprcas9 technology to.
Okita k, ichisaka t, yamanaka s 2007 generation of germlinecompetent induced pluripotent stem cells. An overview of the manufacturing of human induced pluripotent stem cells under cgmps a tissue acquisition program was established, focusing on defining tissue requirements, working with a tissue recovery agency, establishing forms and standard operating procedures, recovering tissue, and donor eligibility determination. Isolation, culture, and functional characterization of human. However, it then took a further 17 years before pluripotent stem cells were. Characterization of humaninduced pluripotent stem cell. Jan 22, 2016 characterization of induced pluripotent stem cell microvesicle genesis, morphology and pluripotent content. Derivation and characterization of induced pluripotent. Currently, the arterial endothelial cells derived from human pluripotent stem cells lack the range of arterialspecific functions in vitro and the protective function for ischemic tissues in vivo. Generation and mirna characterization of equine induced. Pluripotent embryonic 1 stem cells escs originate from the inner cell mass of the blastocyst stage during embryonic development. This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. It is therefore crucial to establish specific, fast and reliable protocols to detect the hallmarks of pluripotency.
Pluripotent embryonic stem cells escs were first cultured from mice in 1981 1, 2. Reprogram reprogram adult cells, such as blood cells, back to a pluripotent state to become induced pluripotent stem cells ipscs somatic and. Inhibitor chir99021 in human pluripotent stem cells. High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry subarna bhattacharya 1, paul w. Phenotypic characterization of toxic compound effects on cardiomyocytes derived from induced pluripotent stem cells introduction cardiotoxicity remains a primary cause for drug attrition during clinical trials and postmarketing1. Characterization of pluripotent stem cells nature protocols. Chapter characterization and criteria of embryonic stem and induced pluripotent stem cells for a dopamine replacement therapy oliver cooper,1, malin parmar, ole isacson neuroregeneration institute, mclean hospital, harvard medical school, harvard stem cell institute, belmont, ma, usa. In order to fully utilize companion animal pluripotent stem cells robust, standardized methods of characterization must be developed to ensure that safe and effective treatments can be delivered. The study provides important insights into the underlying molecular mechanisms that regulate atriallike hipsccm electrophysiology and. Microrna characterization in equine induced pluripotent stem. Antibodybased tools and protocols for characterization of pluripotent stem cells duration. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicityprofiling techniques to characterize mitochondrial function in induced pluripotent stem cell ipsc derived human cardiomyocytes in the presence of differing carbon sources. Catherine m schwartz, mahendra s rao, ernest arenas and mark p mattson.
Characteristics and characterization of human pluripotent. Derivation and characterization of induced pluripotent stem. Characterization of companion animal pluripotent stem cells y. Global characterization of x chromosome inactivation in human pluripotent stem cells shiran bar,1 lev roz seaton,1 uri weissbein,1 talia eldargeva,2,3 and nissim benvenisty1,4, 1the azrieli center for stem cells and genetic research, department of genetics, silberman institute of life sciences, the hebrew university, jerusalem, israel. Induced pluripotent stem cell generation and characterization. Schwartzc, timothy hackerd, vernella vickermana, scott swanson a, ning lenga,e, bao kim nguyen, angela elwell a, jennifer bolina, matthew e. Characterization of induced pluripotent stem cell microvesicle genesis, morphology and pluripotent content.
Culture, expansion, and cryopreservation of pluripotent stem cells in feederfree culture system essential 8 medium psc cryopreservation kit timeline for the generation of an ipsc line the entire reprogramming process from expanded fibroblast culture to stable induced pluripotent stem. Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. Derivation, enrichment and characterization of dopaminergic. The pluripotent stem cell workflow characterize analyze stem cells and their derivatives using a wide range of cellular and molecular characterization methods, as validation is critical in ipsc research. Cell reports report global characterization of x chromosome inactivation in human pluripotent stem cells shiran bar,1 lev roz seaton,1 uri weissbein,1 talia eldargeva,2,3 and nissim benvenisty1,4, 1the azrieli center for stem cells and genetic research, department of genetics, silberman institute of life sciences, the hebrew university, jerusalem, israel. Pdf characterization of cancer stemlike cells derived. Induced pluripotent stem ips cells can be generated by introducing defined factors into somatic cells. Production and characterization of human macrophages from. Nov 30, 2017 comprehensive characterization of pluripotent stem cells thermo fisher scientific. Generation and characterization of induced pluripotent stem.
Global characterization of x chromosome inactivation in. Jul 25, 2017 functional characterization of human pluripotent stem cellderived arterial endothelial cells jue zhang, a lifang chu, a zhonggang hou, a, b michael p. May 02, 2019 pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patientspecific induced pluripotent stem cells ipscs has become possible in multiple species. We have recently described manufacturing of human induced pluripotent stem cells ipsc master cell banks mcb generated by a clinically compliant process using cord blood as a starting material baghbaderani et al.
Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Ten years of progress and promise of induced pluripotent stem. Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple. Jul 25, 2017 generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. Comprehensive characterization of pluripotent stem cells thermo fisher scientific. Generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. Characterization of companion animal pluripotent stem cells. These spermatogoniaderived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline.
Comprehensive characterization of pluripotent stem cells. Figure 2 functional characterization of hipscmono in a micro. Jan 27, 2016 pluripotent stem cells pscs have the ability to selfrenew and, with the right cue, differentiate into a wide variety of cell types representative of the three germ layers. Patientderived ipscs provide new opportunities to develop ex vivo cellular models of psychiatric disorders. They are very multipurpose type cells and can differentiate into all cell types of the body adult stem cells 1. Apr 19, 2018 human embryonic stem cells hescs hold great potential for the treatment of various degenerative diseases. Guest1 abstract pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate. Isolation and characterization of multipotent and pluripotent. Pluripotent stem cells have the ability to differentiate into all of the cells of the adult body. Detailed characterization of human induced pluripotent. Functional characterization of human pluripotent stem cellderived arterial endothelial cells jue zhanga, lifang chua, zhonggang houa,b, michael p. Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesiclesbased intercellular communication.
Proper characterization of ipsderived osteoblasts is. Induced pluripotent stem cells in medicine and biology. Characterization and comparison of osteoblasts derived. Firstly, an essential prerequisite for using hpscs in clinical applications is the maintenance of large. Global characterization of x chromosome inactivation in human. Overall, stem cells can be divided into two groups, embryonic and adult stem cells. Pluripotent stem cells pscs have the ability to selfrenew and, with the right cue, differentiate into a wide variety of cell types representative of the three germ layers. Stem cells is read and written by clinical and basic scientists whose expertise encompasses the rapidly expanding fields of stem and progenitor cell biology. Generation and functional characterization of icell microglia from human induced pluripotent stem cells eugenia jones, deepika rajesh, beatriz c. This is similar to the reported characterization of undifferentiated primate es and parthenogenetic stem cells 7, human es cells, and ntes cells 9, 11, 20 but is different from the previously established pluripotent stem cell lines produced from bovine ivf embryos, which were ssea1 positive 14, 17. In this article, darbar and colleagues identify a mechanism by which raguided differentiation of hipscs generates an atriallike electrophysiologic signature through the downstream regulation of calcium channel gene expression by couptfii. Guest1 abstract pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. We confirmed that bipscderived dopaminergic neurons resemble those.