Characterization and therapeutic application of mesenchymal. 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. 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. 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. Global characterization of x chromosome inactivation in human. Firstly, an essential prerequisite for using hpscs in clinical applications is the maintenance of large. Guest1 abstract pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate. Reprogram reprogram adult cells, such as blood cells, back to a pluripotent state to become induced pluripotent stem cells ipscs somatic and. We confirmed that bipscderived dopaminergic neurons resemble those. 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.
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. To this end, we differentiated baboon fibroblastderived induced pluripotent stem cells bipscs into dopaminergic neurons with the application of specific morphogens and growth factors. 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. 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. They are very multipurpose type cells and can differentiate into all cell types of the body adult stem cells 1. Electrophysiologic characterization of calcium handling in. The characteristics of pluripotent stem cells which make them so ideal for clinical use e. Generation and characterization of induced pluripotent. High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry subarna bhattacharya 1, paul w. Generation and characterization of transgenefree human.
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. 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. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these ipscderived macrophages. Pluripotent hescs have a great ability to undergo unlimited selfrenewal in culture and to differentiate into all cell types in the body. Comprehensive characterization of pluripotent stem cells thermo fisher scientific. A facs analysis of monocytes markers cd14, cd45, and integrins cd49d, cd18, cd29, and cd11b on freshly isolated and. 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.
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. Breton a, sharma r, diaz ac, parham ag, graham a, neil c, et al. However, it then took a further 17 years before pluripotent stem cells were. Characteristics and characterization of human pluripotent. 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. 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. Pluripotent stem cells have the ability to differentiate into all of the cells of the adult body. Generation and functional characterization of monocytes and. Development and characterization of a scaffoldfree 3d. In this manuscript, we describe the detailed characterization of the two ipsc clones generated using this process, including whole. Catherine m schwartz, mahendra s rao, ernest arenas and mark p mattson. Induced pluripotent stem cell generation and characterization. Pdf characterization of cancer stemlike cells derived from. Pdf characterization of cancer stemlike cells derived.
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. 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. 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. 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. Production and characterization of human macrophages from. Generation and characterization of induced pluripotent stem. Apr 19, 2018 human embryonic stem cells hescs hold great potential for the treatment of various degenerative diseases. 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. Jan 10, 20 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.
Characterization and comparison of osteoblasts derived. It has been shown that the epigenetic status, such as the presence of dna methylation and histone modifications, changes dramatically during ips cell generation. 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. 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. Stem cells is read and written by clinical and basic scientists whose expertise encompasses the rapidly expanding fields of stem and progenitor cell biology. Antibodybased tools and protocols for characterization of pluripotent stem cells duration. Generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. Isolation and characterization of pluripotent human. Freitas, sarah burton, christie munn, michael hancock, kwi. High efficiency differentiation of human pluripotent stem.
Characterization of humaninduced pluripotent stem cell. Human embryonic stem cells hescs hold great potential for the treatment of various degenerative diseases. Several reports have documented the derivation of pluripotent cells multipotent germline stem cells from spermatogonial stem cells obtained from the adult mouse testis. 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. Isolation and characterization of multipotent and pluripotent. 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. Nov 30, 2017 comprehensive characterization of pluripotent stem cells thermo fisher scientific. Molecular analysis tools for epigenomic characterization. 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.
Characterization of companion animal pluripotent stem cells. Functional characterization of human pluripotent stem cell. The journey of hesc research is not that smooth, as it has faced several challenges which are limited to not only tumor. Aneuploidy in pluripotent stem cells and implications for. These spermatogoniaderived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. They are also the underlying trigger of many diseases. This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Human cms derived from ipscs were studied in standard 2d culture and as cardiac microtissues mts formed in hanging drops. A bioinformatics assay for pluripotency in human cells. Generation and functional characterization of icell microglia from human induced pluripotent stem cells eugenia jones, deepika rajesh, beatriz c. 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. Methods and protocols highlights the best methods and systems for the entire work flow. Derivation and characterization of induced pluripotent stem cells from equine fibroblasts.
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. Dopaminergic neuron differentiation of human pluripotent stem cells. 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. 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. 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. Schwartzc, timothy hackerd, vernella vickermana, scott swanson a, ning lenga,e, bao kim nguyen, angela elwell a, jennifer bolina, matthew e.
Detailed characterization of human induced pluripotent. 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. Overall, stem cells can be divided into two groups, embryonic and adult stem cells. Pluripotent embryonic stem cells escs were first cultured from mice in 1981 1, 2. Nathanson 2 agnieszka dantoniochronowska 3 angelo arias 1 hiroko matsui 3 roy williams 3 cheryl herrera 2 sol m. Generation and mirna characterization of equine induced. 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. Microrna characterization in equine induced pluripotent. One of these developments is the use of induced pluripotent stem ips cells. Proper characterization of ipsderived osteoblasts is. Patientderived ipscs provide new opportunities to develop ex vivo cellular models of psychiatric disorders.
Here, we combine singlecell rna sequencing and crisprcas9 technology to. 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. 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. Okita k, ichisaka t, yamanaka s 2007 generation of germlinecompetent induced pluripotent stem cells. Ten years of progress and promise of induced pluripotent stem. They may therefore represent a potentially ideal source of homogeneous and highquality msc.
Induced pluripotent stem ips cells can be generated by introducing defined factors into somatic cells. Highthroughput and costeffective characterization of induced pluripotent stem cells author links open overlay panel matteo dantonio 1 grace woodruff 2 jason l. 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. 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. Global characterization of x chromosome inactivation in. Highthroughput and costeffective characterization of. Characterization and comparison of osteoblasts derived from. Derivation and characterization of induced pluripotent. 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. Microrna characterization in equine induced pluripotent stem.
Functional characterization of induced pluripotent stem. Jul 25, 2017 generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. It is therefore crucial to establish specific, fast and reliable protocols to detect the hallmarks of pluripotency. 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. Figure 2 functional characterization of hipscmono in a micro.
Characterization of pluripotent stem cells nature protocols. Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Pluripotent embryonic 1 stem cells escs originate from the inner cell mass of the blastocyst stage during embryonic development. Chapter characterization and criteria of embryonic. Induced pluripotent stem cells in medicine and biology. Pluripotent stem cell characterization methods youtube. Since the first generation of embryonic stem cells escs1,2 and induced pluripotent stem cells ipscs 3,4, numerous cell culture techniques focusing on. 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. Reference maps of human es and ips cell variation enable highthroughput characterization of stem cells. Pluripotent stem cell product guide thermo fisher scientific.
Characterization of monocytes and macrophages derived from human induced pluripotent stem cells xucao,1 francijnae. These cells had the capability to differentiate into derivatives of all three germ layers endoderm, ectoderm, and mesoderm, self. Monocytes and macrophages are essential for immune defense and tissue hemostasis. The study provides important insights into the underlying molecular mechanisms that regulate atriallike hipsccm electrophysiology and. 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. Antibodybased tools and protocols for characterization of pluripotent stem cells. Functional characterization of human pluripotent stem. Takahashi k, yamanaka s 2006 induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
Generation and functional characterization of monocytes. Functional characterization of induced pluripotent stem cell. 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. Derivation and characterization of induced pluripotent stem. Guest1 abstract pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. Characterization of induced pluripotent stem cell microvesicle genesis, morphology and pluripotent content.
Human stem cellderived tissue cells may provide such a model. While it is believed that a significant percentage of cardiovascular diseases are due to environmental. Phenotypic characterization of toxic compound effects on. Jan 22, 2016 characterization of induced pluripotent stem cell microvesicle genesis, morphology and pluripotent content. Isolation, culture, and functional characterization of. Generation and characterization of pluripotent stem cells. Characterization of companion animal pluripotent stem cells y. Isolation, culture, and functional characterization of human. The cells must be shown to be safe for clinical use and not produce tumors or differentiate into unwanted cell types. Ten years of progress and promise of induced pluripotent. 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.