scholarly journals Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale

PeerJ ◽  
2019 ◽  
Vol 6 ◽  
pp. e6143 ◽  
Author(s):  
Xiaoman Wang ◽  
Tingting Liao ◽  
Cong Wan ◽  
Xiaoyu Yang ◽  
Jiexiang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Germ Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Induction System ◽  
Induced Pluripotent ◽  
In Vitro Induction

Background The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. Methods The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. Results In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. Discussion The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.

A Library of Sickle Cell Anemia Induced Pluripotent Stem Cells of Diverse Haplotypes and Ethnicities

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2354-2354
Author(s):  
Seonmi Park ◽  
Andreia Gianotti-Sommer ◽  
David H.K. Chui ◽  
Maria Stella Figueiredo ◽  
Abdulrahman Alsultan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Pluripotent Stem Cells ◽  
Globin Gene ◽  
Genetic Modifiers ◽  
Induced Pluripotent

Abstract The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table. Table 1. Number of subjects recruited to date 98 Number of subjects with iPSC lines established 56 Average number of iPSC lines per subject 3 (total of 158 lines generated) Quality control status of iPSC lines All lines are expanded and banked, mycoplasma free, express pluripotency markers Subjects with target cells differentiated (erythrocytes) 25 Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell. Disclosures No relevant conflicts of interest to declare.

scholarly journals Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells

2017 ◽  
Vol 20 (4) ◽  
pp. 533-546.e7 ◽  
Author(s):  
Christopher DeBoever ◽  
He Li ◽  
David Jakubosky ◽  
Paola Benaglio ◽  
Joaquin Reyna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Genetic Variation ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Induced Pluripotent
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