Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.
Animals ; Cell Culture Techniques ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Human Embryonic Stem Cells ; Humans ; Induced Pluripotent Stem Cells ; Pluripotent Stem Cells ; Stem Cells

Recent advances in stem cell biology have dramatically increased the understanding of molecular and cellular mechanism of pluripotency and cell fate determination. Additionally, pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, arose as essential resources for disease modeling and cellular therapeutics. Despite these advancements, the epigenetic dysregulation in pluripotency such as the imprinting status, and X chromosome dosage compensation, and its consequences on future utility of PSCs yet remain unresolved. In this review, we will focus on the X chromosome regulation in human PSCs (hPSCs). We will introduce the previous findings in the dosage compensation process on mouse model, and make comparison with those of human systems. Particularly, the X chromosome activation status of human preimplantation embryos, and the regulation of the active X chromosome by human specific lincRNA, X Active Coating Transcript (XACT), will be discussed. We will also discuss the recent findings on higher order X chromosome architecture, and abnormal X chromosome status in hPSCs.
Animals ; Biology* ; Blastocyst ; Chromosomes, Human, X* ; Compensation and Redress ; Embryonic Stem Cells ; Epigenomics ; Humans* ; Induced Pluripotent Stem Cells ; Mice ; Pluripotent Stem Cells ; Stem Cells* ; X Chromosome

Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.
Humans ; Infant, Newborn ; Male ; Germ Cells ; Embryonic Stem Cells ; Cell Differentiation ; Infertility, Male ; Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a type of cells similar to embryonic stem cells but produced by reprogramed somatic cells. Through in vitro differentiation of iPSCs, we can interrogate the evolution history as well as the various characteristics of macrophages. iPSCs derived macrophages are not only a good model for drug screening, but also an important approach for immunotherapy. This review summarizes the advances, challenges, and future directions in the field of iPSCs-derived macrophages.
Cell Differentiation ; Embryonic Stem Cells ; Induced Pluripotent Stem Cells ; Macrophages

Blood transfusion is a well-established cell therapy. However, blood available for transfusion is a limited resource and is available only through donations by healthy volunteers. Moreover, the perpetual and widespread shortage of blood products, problems related to transfusion transmitted infections, and new emerging pathogens have elicited an increase in demand for artificial blood. Therefore, research for alternative RBC substitutes has begun in the 1960s. Hemoglobin-based oxygen carriers (HBOC) and perfluorocarbon-based oxygen carrier (PBOC) were two popular study subjects; however, research on these substitute candidates was halted due to unsatisfactory results and safety issues, including death, in the 1990s. Since then, worldwide efforts to produce RBC have shifted over to stem cell-derived RBC production using cord blood and G-CSF-mobilized peripheral blood stem cells, and some progress has been made. In terms of practical usefulness, however, large-scale production and cost effectiveness are still problematic. Recently, human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) have shown the potential to produce RBCs as unlimited cell sources. These two methods using hESCs and hiPSCs are also cost-effective since autologous and O, D negative blood RBCs will be used for alloimmunized patients with multiple alloantibodies or rare blood types (high incidence antigens) as well as universal blood production. We will review the current research on in vitro RBC production from hematopoietic stem cells and pluripotent stem cells and assess future directions in this field.
Blood Substitutes ; Blood Transfusion ; Cell- and Tissue-Based Therapy ; Cost-Benefit Analysis ; Erythrocytes* ; Fetal Blood ; Healthy Volunteers ; Hematopoietic Stem Cells ; Human Embryonic Stem Cells ; Humans ; In Vitro Techniques ; Incidence ; Induced Pluripotent Stem Cells ; Isoantibodies ; Oxygen ; Pluripotent Stem Cells ; Stem Cells*

Pluripotent stem cells can differentiate into many cell types including mature hepatocytes, and can be used in the development of new drugs, treatment of diseases, and in basic research. In this study, we established a protocol leading to efficient hepatic differentiation, and compared the capacity to differentiate into the hepatocyte lineage of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Optimal combinations of cytokines and growth factors were added to embryoid bodies produced by both types of cell. Differentiation of the cells was assessed with optical and electron microscopes, and hepatic-specific transcripts and proteins were detected by quantitative reverse transcription polymerase chain reaction and immunocytochemistry, respectively. Both types of embryoid body produced polygonal hepatocyte-like cells accompanied by time-dependent up regulation of genes for α-fetoprotein, albumin (ALB), asialoglycoprotein1, CK8, CK18, CK19, CYP1A2, and CYP3A4, which are expressed in fetal and adult hepatocytes. Both types of cell displayed functions characteristic of mature hepatocytes such as accumulation of glycogen, secretion of ALB, and uptake of indocyanine green. And these cells are transplanted into mouse model. Our findings indicate that hESCs and hiPSCs have similar abilities to differentiate into hepatocyte in vitro using the protocol developed here, and these cells are transplantable into damaged liver.
Adult ; Animals ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP3A ; Cytokines ; Embryoid Bodies ; Glycogen ; Hepatocytes ; Human Embryonic Stem Cells* ; Humans* ; Immunohistochemistry ; In Vitro Techniques ; Indocyanine Green ; Induced Pluripotent Stem Cells* ; Intercellular Signaling Peptides and Proteins ; Liver ; Mice ; Pluripotent Stem Cells ; Polymerase Chain Reaction ; Reverse Transcription ; Up-Regulation

Animals ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Embryonic Stem Cells ; cytology ; metabolism ; Germ Cells ; cytology ; metabolism ; Germ Layers ; cytology ; metabolism ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice ; Pluripotent Stem Cells ; cytology ; metabolism ; Reproductive Techniques, Assisted

OBJECTIVETo investigate whether mouse-induced pluripotent stem (iPS) cell line IP14D-1 has the potential to differentiate into induced primordial germ cells (iPGCs), and to explore the changes in the expression of iPGCs-differentiation associated genes and their possible mechanisms.

METHODSUndifferentiated IP14D-1 was cultured to proliferate and then differentiated to form 4-, 7- and 9-day-old induced embryoid bodies (iEBs) in vitro, respectively. RT-PCR and immunofluorescence were used to detect the expressions of Lin28, Blimpl, Stra8 and Mvh, as well as the localization of the corresponding protein in iEBs.

RESULTSThe expression of Blimpl was higher than that of Lin28 in the undifferentiated IP14D-1 and mouse embryonic stem cells (mESCs). Mvh and Stra8 as well as mESCs and EBs were also expressed in IP14D-1 and iEBs, but with no significant differences. The expression of Lin28 was gradually increased in the IP14D-1-derived iEBs from 4 to 7 days, but decreased at 9 days, and the expression of Blimp1 was gradually reduced with the prolonged growing time of iEBs.

CONCLUSIONA stable system was established for the culture and differentiation of IP14D-1 and IP14D-1-derived iEBs. The expressions of Lin28, Blimp1, Mvh and Stra8 were not significantly different between the undifferentiated IP14D-1 and mESCs, nor were the expressions of Mvh and Stra8 between iEBs and EBs. IP14D-1 and iEBs had the potential to differentiate into iPGCs, which increased in number in the 7-day-old iEBs, and the expression of iPGC-differentiation associated Lin28 became lower in the older iEBs.

Animals ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; Germ Cells ; cytology ; Induced Pluripotent Stem Cells ; cytology ; Male ; Mice ; Mice, Inbred BALB C

The survival rates of boys and men with cancer have increased due to advances in cancer treatments; however, maintenance of quality of life, including fertility preservation, remains a major issue. Fertile male patients who receive radiation and/or chemotherapy face temporary, long-term, or permanent gonadal damage, particularly with exposure to alkylating agents and whole-body irradiation, which sometimes induce critical germ cell damage. These cytotoxic treatments have a significant impact on a patient's ability to have their own biological offspring, which is of particular concern to cancer patients of reproductive age. Therefore, various strategies are needed in order to preserve male fertility. Sperm cryopreservation is an effective method for preserving spermatozoa. Advances have also been achieved in pre-pubertal germ cell storage and research to generate differentiated male germ cells from various types of stem cells, including embryonic stem cells, induced pluripotent stem cells, and spermatogonial stem cells. These approaches offer hope to many patients in whom germ cell loss is associated with sterility, but are still experimental and preliminary. This review examines the current understanding of the effects of chemotherapy and radiation on male fertility.
Alkylating Agents ; Cryopreservation ; Drug Therapy ; Embryonic Stem Cells ; Fertility ; Fertility Preservation ; Germ Cells ; Gonads ; Hope ; Humans ; Induced Pluripotent Stem Cells ; Infertility ; Infertility, Male ; Male ; Methods ; Quality of Life ; Radiotherapy ; Spermatogenesis ; Spermatozoa ; Stem Cells ; Survival Rate ; Whole-Body Irradiation

To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.
Blood Substitutes ; Cost-Benefit Analysis ; Embryonic Stem Cells ; Erythrocytes* ; Fetal Blood ; Induced Pluripotent Stem Cells ; Stem Cell Research ; Stem Cells*