Derivation Of Embryonic Germ Cells Research Articles

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells

Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.

Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs.

Culture of Mouse Amniotic Fluid-Derived Cells on Irradiated STO Feeders Results in the Generation of Primitive Endoderm Cell Lines Capable of Self-Renewal in vitro

The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies.

Induction of pluripotency in primordial germ cells.

Primordial germ cells (PGCs) are the founder cells of all gametes. PGCs differentiate from pluripotent epiblasts cells by mesodermal induction signals during gastrulation. Although PGCs are unipotent cells that eventually differentiate into only sperm or oocytes, they dedifferentitate to pluripotent stem cells known as embryonic germ cells (EGCs) in vitro and give rise to testicular teratomas in vivo, which indicates a "metastable" differentiation state of PGCs. We have shown that an appropriate level of phosphoinositide-3 kinase (PI3K)/Akt signaling, balanced by positive and negative regulators, ensures the establishment of the male germ lineage by preventing its dedifferentiation. Specifically, hyper-activation of the signal leads to testicular teratomas and enhances EGC derivation efficiency. In addition, PI3K/Akt signaling promotes PGC dedifferentiation via inhibition of the tumor suppressor p53, a downstream molecule of the PI3K/Akt signal. On the other hand, Akt activation during mesodermal differentiation of embryonic stem cells (ESCs) generates PGC-like pluripotent cells, a process presumably induced through equilibrium between mesodermal differentiation signals and dedifferentiation-inducing activity of Akt. The transfer of these cells to ESC culture conditions results in reversion to an ESC-like state. The interconversion between ESC and PGC-like cells helps us to understand the metastability of PGCs. The regulatory mechanisms of PGC dedifferentiation are discussed in comparison with those involved in the dedifferentiation of testicular stem cells, ESC pluripotency, and somatic nuclear reprogramming.

Epigenetic reprogramming in the porcine germ line

BackgroundEpigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.ResultsOur results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.ConclusionsOur study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.

Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

Engineering Musculoskeletal Tissues with Human Embryonic Germ Cell Derivatives

The cells derived from differentiating embryoid bodies of human embryonic germ (hEG) cells express a broad spectrum of gene markers and have been induced toward ecto- and endodermal lineages. We describe here in vitro and in vivo differentiation of hEG-derived cells (LVEC line) toward mesenchymal tissues. The LVEC cells express many surface marker proteins characteristic of mesenchymal stem cells and differentiated into cartilage, bone, and fat. Homogenous hyaline cartilage was generated from cells after 63 population doublings. In vivo results demonstrate cell survival, differentiation, and tissue formation. The high proliferative capacity of hEG-derived cells and their ability to differentiate and form three-dimensional mesenchymal tissues without teratoma formation underscores their significant potential for regenerative medicine. The adopted coculture system also provides new insights into how a microenvironment comprised of extracellular and cellular components may be harnessed to generate hierarchically complex tissues from pluripotent cells.

Derivation of Embryonic Germ Cells from Post Migratory Primordial Germ Cells, and Methylation Analysis of Their Imprinted Genes by Bisulfite Genomic Sequencing

Derivation of Embryonic Germ Cells from Post Migratory Primordial Germ Cells, and Methylation Analysis of Their Imprinted Genes by Bisulfite Genomic Sequencing

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.

Expression of pluripotent stem cell markers in the human fetal ovary

Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro. Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad. These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.

Collection and culture of primordial germ cells from cynomolgus monkeys (Macaca fascicularis).

Aim: To clarify the location of primordial germ cells (PGC) in an embryo of target-age and to examine the culture environment of the PCG. Methods: The days of ovulation and fertilization were estimated by measuring the serum concentration of estrogen. Pregnancy was confirmed by measurement of the serum concentration of the beta subunit of macaque chorionic gonadotropin and by ultrasonography. We also examined the location of PGC in the embryo at the time of retrieval. Results: Results showed that PGC in an embryo were in the hindguts at day 30 postfertilization, arrived at the genital ridges via mesenteries at approximately day 33 postfertilization, and colonized the gonads by day 36 postfertilization. Conclusions: In conclusion, embryos collected on day 33 postfertilization are more suitable for obtaining PGC from cynomolgus monkeys. The PGC collected from cynomolgus monkey fetuses were cultured under conditions for the derivation and culture of human embryonic germ cells; enzymatically dispersed single cells were cultured on a SIM thioguanine-resistant ouabain-resistant cells (STO) feeder layer with recombinant human leukemia inhibitory factor, recombinant human basic fibroblast growth factor and forskolin. The cells from genital ridges and mesenteries at day 33 postfertilization had alkaline phosphatase (ALP) activity in vitro for a maximum of 13days. In contrast, ALP activity had been held for 2months under the same culture condition when the cells were derived from the gonads at day 66 postfertilization. Derivation of an embryonic germ cell from a cynomolgus monkey was not achieved from these cultures. (Reprod Med Biol 2007; 6: 203-210).

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and β-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

Enhanced extracellular matrix production and differentiation of human embryonic germ cell derivatives in biodegradable poly(ε-caprolactone-co-ethyl ethylene phosphate) scaffold

Extracellular environment regulates cell behavior and also influences the differentiation of stem cells. Two cell lines of pluriopotent human embryonic germ cell derivatives (EBD cells) were cultured on a biodegradable poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) and non-degradable cellulose acetate scaffold. Their cell behaviors including proliferation, differentiation, cell distribution and extracellular matrix production were studied for 4 weeks and 10 months. The proliferation of the EBD cells was enhanced in both of the three-dimensional scaffolds in the first 5 weeks of culture, regardless of the material difference, compared to monolayer culture. While the gene expression profile remained multilineage for the EBD cells cultured in the cellulose acetate fibrous scaffold, much of the neuronal lineage markers were down-regulated in EBD cells cultured in the PCLEEP scaffold. On the other hand, extracellular matrix production was significantly enhanced in the PCLEEP scaffold. The study showed that the polymer substrate could influence the differentiation and growth of pluripotent stem cells in the absence of exogenous biochemical signals.

Proliferation and differentiation of human embryonic germ cell derivatives in bioactive polymeric fibrous scaffold

Human embryonic germ cell derivatives, a heterogeneous population of uncommitted embryoid body derived (EBD) cells, were studied in a bioactive three-dimensional (3D) fibrous culture. Their proliferation, morphology, gene expression and differentiation were investigated to gain insights on development of 3D bioactive scaffold for pluripotent stem cells. The expansion of the EBD cells in 3D environment was significantly higher than their two-dimensional controls after 21 days. No apparent differentiation of the EBD cells cultured in the 3D environment, as indicated by histology and gene expression profile analysis, was evident. Extracellular matrix production was weak in the long-term 3D culture, and the EBD cells maintained their multilineage gene expressions for the period studied. When nerve growth factor (NGF) was surface-immobilized on the fibrous scaffold via chemically-modified Pluronic, the EBD cells cultured in this scaffold showed evidence of entering the neural pathway. An upregulation of tyrosine hydroxylase mRNA expression was observed when EBD cells were cultured in the NGF-immobilized fibrous scaffold, as demonstrated by real-time PCR and immunofluorescence staining. The study suggests the value of such fibrous 3D culture in manipulating stem cell proliferation/differentiation and as a model for developing a bioactive scaffold.

Derivation of embryonic germ cells and male gametes from embryonic stem cells.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Human Embryonic Germ Cell Derivatives Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [>70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.

Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro.

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [>70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.

Physiology of nerve growth factor.

Physiology of nerve growth factor.