Stem Cell Clinic

Embryonic stem cell research – alsa.org

Overview

Stem cells have the ability to divide for indefinite periods in culture and give rise to multiple specialized cell types. They can develop into blood, neurons, bone, muscle, skin and other cell types. They have emerged as a major tool for research into the causes of ALS, and in the search of new treatments.

Types of Stem Cells:

The field of stem cell research is progressing rapidly, and The ALS Association is spearheading work on several critical fronts. The research portfolio supports innovative projects using IPSCs for drug development and disease modeling. The Association is supporting an IPSC core at Cedars-Sinai Medical Center providing access to lines for researchers globally. Several of the big data initiatives are collecting skin cells or blood for IPSC generation, such as Genomic Translation for ALS Clinical Care (GTAC), Project MinE, NeuroLINCS and Answer ALS. The ALS Association also sponsors pre-clinical studies and pilot clinical trials using stem cell transplant approaches to develop the necessary tools for stem cell transplant studies and to improve methods for safety and efficiency. We also support studies that involve isolating IPSCs to develop biomarkers for clinical trials through ALS ACT. In addition, the retigabine clinical trial that we sponsor uses iPSCs derived from participants in parallel with clinical data to help test whether the drug has the desired effect.

Stem cells are being used in many laboratories today for research into the causes of and treatments for ALS. Most commonly, researchers use iPSCs to make a unique source of motor neurons from individual ALS patients to try to understand why and how motor neurons die in ALS. Two types of motor neurons are affected in ALS are upper coriticospinal motor neurons, that when damaged, cause muscle spasticity (uncontrolled movement), and lower motor neurons, that when damaged, cause muscle weakness. Both types can be made from iPSCs to cover the range of pathology and symptoms found in ALS. Astrocytes, a type of support cell, called glia, of the central nervous system (CNS), are also being generated from iPSCs. It is well established that glia play a role in disease process and contribute to motor neuron death.

Motor neurons created from iPSCs have many uses. The availability of large numbers of identical neurons, made possible by iPSCs, has dramatically expanded the ability to search for new treatments. For example, they can also be used to screen for drugs that can alter the disease process. Motor neurons derived from iPSCs can be genetically modified to produce colored fluorescent markers that allow clear visualization under a microscope. The health of individual motor neurons can be tracked over time to understand if a test compound has a positive or negative effect.

Because iPSCs can be made from skin samples or blood of any person, researchers have begun to make cell lines derived from dozens of individuals with ALS. One advantage of iPSCs are that they capture a persons exact genetic material and provide an unlimited supply of cells that can be studied in a dish, which is like persons own avatar. Comparing the motor neurons derived from these cells lines allows them to ask what is common, and what is unique, about each case of ALS, leading to further understanding of the disease process. They are also used to correlate patients clinical parameters, such as site of onset and severity with any changes in the same patients motor neurons.

Stem cells may also have a role to play in treating the disease. The most likely application may be to use stem cells or cells derived from them to deliver growth factors or protective molecules to motor neurons in the spinal cord. Clinical trials of such stem cell transplants are in the early stages, but appear to be safe. In addition, transplantation of healthy astrocytes have the potential to be beneficial in supporting motor neurons in the brain and spinal cord.

While the idea of replacing dying motor neurons with new ones derived from stem cells is appealing, using stem cells as a delivery tool to provide trophic factors to motor neurons is a more realistic and feasible approach. The significant challenge to replacing dying motor neurons is making the appropriate connections between muscles and surrounding neurons.

Isolation of IPSCs from people with ALS in clinical trials is extremely valuable for the identification of unique signatures in the presence or absence of a specific treatment approach and as a read out to test whether a drug or test compound has an impact on the health of motor neurons and/or astrocytes. A positive result gives researchers confidence to move forward to more advanced clinical trials. For example, The ALS Association is currently funding a clinical trial to test the effects of retigabine on motor neurons, which use the enrolled patients individual iPSCs lines derived from collected skin samples and testing whether there is a change in the excitability of motor neurons in people with ALS. (see above).

See Stem Cells Grants See all Scientific Focus Areas View Glossary of Terms

Visit link:
Embryonic stem cell research - alsa.org

Cloning/Embryonic Stem Cells – National Human Genome Research …

Cloning/Embryonic Stem Cells

The term cloning is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases, isolated genes or cells are duplicated for scientific study, and no new animal results. The experiment that led to the cloning of Dolly the sheep in 1997 was different: It used a cloning technique called somatic cell nuclear transfer and resulted in an animal that was a genetic twin -- although delayed in time -- of an adult sheep. This technique can also be used to produce an embryo from which cells called embryonic stem (ES) cells could be extracted to use in research into potential therapies for a wide variety of diseases.

Thus, in the past five years, much of the scientific and ethical debate about somatic cell nuclear transfer has focused on its two potential applications: 1) for reproductive purposes, i.e., to produce a child, or 2) for producing a source of ES cells for research.

The technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. In the simplest terms, the technique used to produce Dolly the sheep - somatic cell nuclear transplantation cloning - involves removing the nucleus of an egg and replacing it with the diploid nucleus of a somatic cell. Unlike sexual reproduction, during which a new organism is formed when the genetic material of the egg and sperm fuse, in nuclear transplantation cloning there is a single genetic "parent." This technique also differs from previous cloning techniques because it does not involve an existing embryo. Dolly is different because she is not genetically unique; when born she was genetically identical to an existing six-year-old ewe. Although the birth of Dolly was lauded as a success, in fact, the procedure has not been perfected and it is not yet clear whether Dolly will remain healthy or whether she is already experiencing subtle problems that might lead to serious diseases. Thus, the prospect of applying this technique in humans is troubling for scientific and safety reasons in addition to a variety of ethical reasons related to our ideas about the natural ordering of family and successive generations.

Several important concerns remain about the science and safety of nuclear transfer cloning using adult cells as the source of nuclei. To date, five mammalian species -- sheep, cattle, pigs, goats, and mice -- have been used extensively in reproductive cloning studies. Data from these experiments illustrate the problems involved. Typically, very few cloning attempts are successful. Many cloned animals die in utero, even at late stages or soon after birth, and those that survive frequently exhibit severe birth defects. In addition, female animals carrying cloned fetuses may face serious risks, including death from cloning-related complications.

An additional concern focuses on whether cellular aging will affect the ability of somatic cell nuclei to program normal development. As somatic cells divide they progressively age, and there is normally a defined number of cell divisions that can occur before senescence. Thus, the health effects for the resulting liveborn, having been created with an "aged" nucleus, are unknown. Recently it was reported that Dolly has arthritis, although it is not yet clear whether the five-and-a-half-year-old sheep is suffering from the condition as a result of the cloning process. And, scientists in Tokyo have shown that cloned mice die significantly earlier than those that are naturally conceived, raising an additional concern that the mutations that accumulate in somatic cells might affect nuclear transfer efficiency and lead to cancer and other diseases in offspring. Researchers working with clones of a Holstein cow say genetic programming errors may explain why so many cloned animals die, either as fetuses or newborns.

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered on the misperception that a child or many children could be produced who would be identical to an already existing person. This fear is based on the idea of "genetic determinism" -- that genes alone determine all aspects of an individual -- and reflects the belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which he or she develops. Nonetheless, many of the concerns about cloning have focused on issues related to "playing God," interfering with the natural order of life, and somehow robbing a future individual of the right to a unique identity.

Several groups have concluded that reproductive cloning of human beings creates ethical and scientific risks that society should not tolerate. In 1997, the National Bioethics Advisory Commission recommended that it was morally unacceptable to attempt to create a child using somatic cell nuclear transfer cloning and suggested that a moratorium be imposed until safety of this technique could be assessed. The commission also cautioned against preempting the use of cloning technology for purposes unrelated to producing a liveborn child.

Similarly, in 2001 the National Academy of Sciences issued a report stating that the United States should ban human reproductive cloning aimed at creating a child because experience with reproductive cloning in animals suggests that the process would be dangerous for the woman, the fetus, and the newborn, and would likely fail. The report recommended that the proposed ban on human cloning should be reviewed within five years, but that it should be reconsidered "only if a new scientific review indicates that the procedures are likely to be safe and effective, and if a broad national dialogue on societal, religious and ethical issues suggests that reconsideration is warranted." The panel concluded that the scientific and medical considerations that justify a ban on human reproductive cloning at this time do not apply to nuclear transplantation to produce stem cells. Several other scientific and medical groups also have stated their opposition to the use of cloning for the purpose of producing a child.

The cloning debate was reopened with a new twist late in 1998, when two scientific reports were published regarding the successful isolation of human stem cells. Stem cells are unique and essential cells found in animals that are capable of continually reproducing themselves and renewing tissue throughout an individual organism's life. ES cells are the most versatile of all stem cells because they are less differentiated, or committed, to a particular function than adult stem cells. These cells have offered hope of new cures to debilitating and even fatal illness. Recent studies in mice and other animals have shown that ES cells can reduce symptoms of Parkinson's disease in mouse models, and work in other animal models and disease areas seems promising.

In the 1998 reports, ES cells were derived from in vitro embryos six to seven days old destined to be discarded by couples undergoing infertility treatments, and embryonic germ (EG) cells were obtained from cadaveric fetal tissue following elective abortion. A third report, appearing in the New York Times, claimed that a Massachusetts biotechnology company had fused a human cell with an enucleated cow egg, creating a hybrid clone that failed to progress beyond an early stage of development. This announcement served as a reminder that ES cells also could be derived from embryos created through somatic cell nuclear transfer, or cloning. In fact, several scientists believed that deriving ES cells in this manner is the most promising approach to developing treatments because the condition of in vitro fertilization (IVF) embryos stored over time is questionable and this type of cloning could overcome graft-host responses if resulting therapies were developed from the recipient's own DNA.

For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical.

Since 1996, Congress has prohibited researchers from using federal funds for human embryo research. In 1999, DHHS announced that it intended to fund research on human ES cells derived from embryos remaining after infertility treatments. This decision was based on an interpretation "that human embryonic stem cells are not a human embryo within the statutory definition" because "the cells do not have the capacity to develop into a human being even if transferred to the uterus, thus their destruction in the course of research would not constitute the destruction of an embryo." DHHS did not intend to fund research using stem cells derived from embryos created through cloning, although such efforts would be legal in the private sector.

In July 2001, the House of Representatives voted 265 to 162 to make any human cloning a criminal offense, including cloning to create an embryo for derivation of stem cells rather than to produce a child. In August 2002, President Bush, contending with a DHHS decision made during the Clinton administration, stated in a prime-time television address that federal support would be provided for research using a limited number of stem cell colonies already in existence (derived from leftover IVF embryos). Current bills before Congress would ban all forms of cloning outright, prohibit cloning for reproductive purposes, and impose a moratorium on cloning to derive stem cells for research, or prohibit cloning for reproductive purposes while allowing cloning for therapeutic purposes to go forward. As of late June, the Senate has taken no action. President Bush's Bioethics Council is expected to recommend the prohibition of reproductive cloning and a moratorium on therapeutic cloning later this summer.

Prepared by Kathi E. Hanna, M.S., Ph.D., Science and Health Policy Consultant

Last Reviewed: April 2006

Continue reading here:
Cloning/Embryonic Stem Cells - National Human Genome Research ...

Pros and Cons of Stem Cell Research – thebalance.com

Debates over the ethics of embryonic stem cell research continue to divide scientists, politicians and religious groups. However, promising developments in other areas of stem cell research might lead to solutions that bypass these ethical issues. These new developments could help win stem cell research more support from those against embryonic stem cell research, since they don't require the destruction of blastocysts.

Latest Developments

The most recent research has shown that there are many options available other than working with embryonic stem cells. Stem cells can be obtained from cord blood or derived by manipulating differentiated cells (i.e. skin cells) to revert them to a pluripotent state. These are alternatives that may help broaden the acceptance of stem cell research.

Background

In November 1998 the first published research paper reported that stem cells could be taken from human embryos. Subsequent research led to the ability to maintain undifferentiated stem cell lines (pluripotent cells) and techniques for differentiating them into cells specific to various tissues and organs.

The debates over the ethics of stem cell research began almost immediately in 1999, despite reports that stem cells cannot grow into complete organisms.

In 2000 2001, governments worldwide were beginning to draft proposals and guidelines in an effort to control stem cell research and the handling of embryonic tissues, and reach universal policies to prevent brain-drains (emigration of top scientists) between countries.

The CIHR (Canadian Institute of Health Sciences) drafted a list of recommendations for stem cell research in 2001. The Clinton administration drafted guidelines for stem cell research in 2000, but Clinton left office prior to them being released. The Bush government has had to deal with the issue throughout his administration.

Australia, Germany, UK and other countries have also formulated policies.

Pros

The excitement about stem cell research is primarily due to the medical benefits in areas of regenerative medicine and therapeutic cloning. Stem cells provide huge potential for finding treatments and cures to a vast array of diseases including different cancers, diabetes, spinal cord injuries, Alzheimers, MS, Huntingtons, Parkinsons and more.

There is endless potential for scientists to learn about human growth and cell development from studying stem cells.

Use of adult-derived stem cells, from blood, cord blood, skin and other tissues, known as IPSCs, has been demonstrated to be effective for treating different diseases in animal models. Umbilical-cord-derived stem cells (obtained from the cord blood) have also been isolated and utilized for various experimental treatments. Another option is use of uniparental stem cells. Although these cells lines have some disadvantages or shortcomings compared to embryonic cell lines (they are shorter-lived), there is vast potential if enough money is invested in researching them further, and they are not technically considered individual living beings by pro-life advocates.

Cons

Use of embryonic stem cells for reasearch involves the destruction of blastocysts formed from laboratory-fertilized human eggs. For those who believe that life begins at conception, the blastocyst is a human life and to destroy it is unacceptable and immoral.

This seems to be the only controversial issue standing in the way of stem cell research in North America.

Where It Stands

In the summer of 2006 President Bush stood his ground on the issue of stem cell research and vetoed a bill passed by the Senate that would have expanded federal funding of embryonic stem cell research.

Currently, American federal funding can only go to research on stem cells from existing (already destroyed) embryos. Similarly, in Canada, as of 2002, scientists cannot create or clone embryos for research but must used existing embryos discarded by couples. The UK allows embryonic stem cell cloning.

Use of stem cell lines from alternative non-embryonic sources has received more attention in recent years and has already been demonstrated as a successful option for treatment of certain diseases. For example, adult stem cells can be used to replace blood-cell-forming cells killed during chemotherapy in bone marrow transplant patients. Biotech companies such as Revivicor and ACT are researching techniques for cellular reprogramming of adult cells, use of amnionic fluid, or stem cell extraction techniques that do not damage the embryo, that also provide alternatives for obtaining viable stem cell lines.

Out of necessity, the research on these alternatives is catching up with embryonic stem cell research and, with sufficient funding, other solutions might be found that are acceptable to everyone.

On March 9, 2009, President Obama overturned Bush's ruling, allowing US Federal funding to go to embryonic stem cell research. However, the stipulation applies that normal NIH policies on data sharing must be followed. Despite the progress being made in other areas of stem cell research, using pluripotent cells from other sources, many American scientists were putting pressure on the government to allow their participation and compete with the Europeans. However, many people are still strongly opposed.

What do YOU think? Take the poll and see what other people voted:

Are you FOR or AGAINST embryonic stem cell research?

View Results

2016 About, Inc. All rights reserved.

View original post here:
Pros and Cons of Stem Cell Research - thebalance.com

Embryonic Stem Cells and the Germ Cell Lineage | InTechOpen

2.1. Primordial germ cell specification and candidate genes controlling germline establishment

The establishment of early germ cells and their successful maturation are complex processes, and require frequent changes in physiology, location and transcriptional profile of involved cells. Germline establishment in mammals occurs via inductive signaling, in contrast to lower organisms such as flies and worms where the germ cell identity is transmitted via the inheritance of germ plasm (McLaren, 1999, 2000; Saitou et al., 2002). In rodents and humans, the first glimpses of primordial germ cell (PGC) formation are observed in the embryo after implantation and gastrulation, when the epiblast, endoderm, mesoderm and ectoderm are first established (Matsui & Okamura, 2005). At this time, in response to molecular cues including Bone Morphogenetic Proteins (BMP4 and BMP8a) from the yolk sac, a population of pluripotent stem cells is segregated from the ICM and set physically apart from the extra-embryonic ectoderm or yolk sac of the embryo (Figure 1) (Lawson et al., 1999; Ying 2000, 2001). While BMPs provide the inductive signal to epiblast stem cells to supply PGCs, it is not clear what signals control the size of the PGC founder population or what molecules signal the termination of PGC specification. BMPs are crucial to specification of PGCs due to their activation of the ALK2 receptor and Smad-1/5 signaling pathways, as evidenced in mice (Hayashi et al., 2002). In the human embryo, the effects of BMPs on germline specification are unclear because there is very limited access to early embryonic samples. From work in our laboratory, it appears that similar BMP protein pathways are activated in the human embryo during gastrulation that direct PGC specification (Clark & Reijo Pera, 2006, Kee et al, 2006).

Additional mechanisms that may assist germline specification are the activation of pathways that either promote PGC survival and/or inhibit molecules that promote somatic differentiation of PGCs (Ewen & Koopman, 2010). As such, mouse and human PGCs retain expression of several biomarkers of pluripotency, including Oct4, Nanog and Sox2 which underlies their close resemblance to other pluripotent stem cells (Clark & Reijo Pera, 2006; Medrano et al., 2010; Nicholas et al., 2009). Interestingly, the transcription factor Sox2 is expressed on both mouse and human embryonic stem cells but unlike in the mouse, human Sox2 expression is diminished when PGCs migrate to the fetal gonad (Perrett et al., 2008). Coincident with the timing of germline specification in the mouse (E7.5), PGCs near the extraembryonic ectoderm begin to express germ-cell specific markers such as Blimp-1 (Prdm1 in human), Stella (Dppa3 in human), E-Cadherin, and Dazl and harbor alkaline phosphatase activity. Blimp-1/Prdm-1 is a transcriptional repressor whose activity is restricted to the germ lineage and appears to be critical for maintaining a PGC fate. There is strong evidence to suggest that in the mouse, Blimp-1 actively represses the somatic fate of PGCs by inhibiting expression of key somatic regulators during development (Ohinata et al., 2005; Hayashi et al., 2007). It is unclear whether Prdm-1 carries out a similar function in human germline formation. Once cell-cell communication has been established, the Fragilis (IFITIM-1 in human) and Stella/ Dppa3 genes promote further development of PGCs and may do so in a similar fashion as Blimp-1 in mice (Saitou et al., 2002).

Migration of germ cells to the gonad begins at E8.5 in the mouse and during weeks 4 to 6 of human gestation (first trimester). At this stage, PGCs accumulating at the base of the allantois exit the extraembryonic ectoderm and begin migration to the developing gonads, also known as the genital ridge. During migration, PGCs also proliferate by undergoing mitosis and express a new set of biomarkers, including the CXCR4 receptor in mice and the proto-oncogene c-KIT and its ligand, KIT in both mice and humans (Molyneaux & Wylie, 2004; Gomperts et al., 1994). The DAZ gene homologue, DAZL is also expressed on migrating PGCs. In the mouse, migratory PGCs display pseudopodia that may assist in movement through the hindgut and it is plausible that human germ cells behave similarly since they are also observed in the hindgut during migration. Various somatic tissues interact with PGCs during the migratory path to the gonad. It is likely therefore, that these tissues express molecules and factors that guide or cue the PGCs and help maintain their survival. In the mouse, several candidate molecules have been identified, including receptors such as -1 Integrin and extracellular matrix components such as Collagen I (Chuva de Sousa Lopes et al., 2005; De Felici et al., 2005). Although migration of PGCs is less well understood in human development, germ cells have been histologically observed during the late first trimester, when they undergo migration to the hindgut (Fujimoto et al., 1977; Gaskell et al., 2004; Goto et al., 2004). Male and female PGCs have been isolated from

Developmental Cycle of Mammalian Germ Cells. Life cycle of the mouse and human embryo following fertilization, progressing through gastrulation and producing the germline. The germline develops in the gonads and transmits genetic information to the next generation, thus completing the cycle. Fertilization of oocytes by sperm promotes the formation of a 1-cell zygote that undergoes cell division and cleavage to form a blastocyst. The outer layer of blastocyst gives rise to the trophectoderm while the inner cell mass (ICM) contains embryonic stem cells (ESCs). During gastrulation (E7.5 in mouse; Day 15+ in human), the blastocyst cavitates and develops the three germ layers and the epiblast. The primordial germ cells (PGCs) are specified and localize near the extra-embryonic ectoderm, at the base of the allantois. Once PGCs are specified, they migrate to the fetal gonads and undergo sex-specific developmental to male and female gonocytes. Subsequently, male gonocytes undergo spermatogenesis while female gonocytes enter meiotic prophase I and begin oogenesis. Adapted from Schuh-Huerta et al., 2011.

10-week old fetuses and were observed to express Alkaline Phosphatase (AP), a marker of PGCs (Goto et al., 2004). The morphology of human PGCs also resemble the rounded shape of mouse PGCs. Female-specific germ cells have also been visualized at the ultrastructural level during gonadal development in human fetuses (Motta et al., 1997). Finally, recent studies by Kerr et al. with human fetal gonads (testis and ovary) provide a detailed analysis of pluripotency and germ cell-specific markers. The germ cells of the fetal testis are Oct4 +/Nanog +/ c-Kit+ from week 7 to 15 after which these cells become localized to the testis periphery. Meanwhile, the presumptive gonocytes in the week 15 testis show strong expression of Pumilio2 (PUM2), VASA and DAZL and express low to no pluripotency markers (Oct4, Nanog, c-Kit, Tra1-60, Tra1-81) (Kerr et al., 2007). In the human fetal ovary, as in the testis, the expression of pluripotency markers peaks by week 8 and then declines after week 9, as oocytes enter meiosis (Kerr et al., 2008). Interestingly, the cell surface markers SSEA-1 and SSEA-4 are co-expressed on the female germ cells from week 5 onwards although only SSEA-1 is restricted to the germ cell lineage.

Upon arrival at the genital ridge, germ cells express another germ-cell specific marker, VASA, a cytoplasmic protein that is implicated in translational regulation. The gene encoding VASA expression, DDX4 (Mvh in mouse) is highly conserved among species and is expressed exclusively in both male and female pre-meiotic germ cells (Gustafson & Wessel, 2010). This finding underlines the importance of the VASA protein in germline function and makes it an attractive candidate for further study. Along with VASA, other factors produced are germ cell nuclear antigen-1 (GCNA-1) and E-cadherin. The sex-specific character of the developing gonad is controlled by the chromosomal constitution of gonadal somatic cells. In particular, the SRY gene expressed on the Y chromosome in mammals is thought to be an essential regulator of various downstream targets including the Sox9 gene that controls male gonadal development (McLaren, 1995, 2003). Once within the gonad, germ cells associate with Sertoli cells to form testis cords and this interaction induces the expression of VASA in post-migratory PGCs. VASA expression is induced in both male and female PGCs and persists until these cells enter meiosis and after which its levels diminish (Castrillon et al., 2000; Toyooka et al., 2000).

During germline development, an extensive remodeling of the epigenetic landscape occurs. This takes place during embryogenesis and during PGC migration to the gonad (Nicholas et al, 2009). The first wave of epigenetic remodeling occurs during implantation of the blastocyst and involves the erasure of all DNA methylation at CpG islands except those at imprinted gene loci. This transition is observed in all cells of the embryo, including the primitive germline. In female PGCs, there is another level of epigenetic change in the form of random X inactivation wherein one copy of the X chromosome pair is silenced. The second wave of epigenetic remodeling occurs when PGCs migrate to the primitive gonads and their paternal or maternal imprinted loci undergo a gradual process of erasure (Hajkova et al., 2002; Yamazaki et al., 2003). This phase occurs only in germ cells and may help to prime them for sex-specific DNA remethylation, when their developmental programs are established (Durcova-Hills et al., 2006). In the mouse, the re-establishment of imprints takes place prior to birth in the male prospermatogonia (E15) and only after birth in oocytes (Lucifero et al., 2002). In addition to DNA methylation changes, male and female germ cells also undergo post-translational histone modifications and RNA-mediated silencing (Reviewed in Tasler, 2009 & Nicolas et al., 2009).

The successful passage of germ cells through meiosis is a unique and highly rigorous process. However, between male and female embryos, the timing of meiosis is different. Male germ cells are restricted from entering meiosis while female germ cells enter meiosis within the embryo. Although the mechanisms for these contrasting behaviors are unclear, it appears that the gonadal cells provide the signal for (or against) meiotic entry (Brennan & Capel, 2004; Ewen & Koopman, 2010). One signal could be retinoic acid (RA) produced in the fetal ovary, which in turn induces Stra8, a key regulator of meiotic entry. In female germ cells destined to become oocytes, mitotic divisions cease and meiotic prophase begins with the correct stimuli (Borum, 1961); eventually oocytes arrest during Meiosis I prior to fetal birth and will only resume meiosis upon receiving hormonal signals during adulthood (Peters, 1970). Meanwhile, male germ cells transition from primordial status to the gonocyte stage, stop proliferating and remain quiescent in the fetal seminiferous tubules until after birth. The post-natal gonocytes then commit to a spermatogonial stem cell (SSC) fate and amplify through self-renewal or enter meiosis to initiate spermatogenesis. In both male and female germ cells, the synaptonemal complex proteins (SCPs) SCP-1, SCP-2 and SCP-3 are critical components of the meiotic machinery during chromosomal segregation (Chuma et al., 2001; Parra et al., 2004; Yuan et al., 2000). The completion of meiosis signals that germ cells have matured into haploid male and female gametes. At this stage, oocytes exclusively express GDF9 and spermatocytes express TEKT1. However, one feature that distinguishes human and mouse germline differentiation is the synchronization of meiotic entry, as in human fetal gonads, one can observe both pre-meiotic and meiotic germ cells in close proximity (Anderson, 2007).

Significant efforts have been made to culture mouse and human PGCs and gonocyes in vitro. In the case of mouse germ cells, the addition of endogenous factors known to affect germ cell development such as BMPs, RA, LIF and Forskolin have produced mixed results in maintaining PGCs in culture. For example, adding LIF enhanced PGC survival but the observations with the use of other factors not as clear. PGCs may also behave erratically in culture (showing low survival rates and non sex-specific behavior) because of the lack of a normal somatic environment (Childs et al., 2008). Studies of PGCs by Shamblott et al., Turnpenny et al. and Tu et al. with fetal human gonocytes resulted in a mixture of cellular phenotypes. Some gonocytes appeared rounded while others took on an elongated or spindly appearance. In addition, they appeared to have different proliferation rates (Shamblott et al., 1998; Turnpenny et al., 2003; Tu et al., 2007). Currently, it is not at all clear that these cells resemble their germ cell counterparts in vivo, but improvements in culture conditions and the cellular microenvironment will certainly help in this regard.

As delineated earlier, mammalian germ cells populate the testis and ovary during development in a incredibly dynamic manner. During early prenatal mice and human embryo development, PGCs migrate to the primitive gonad (gonadal ridge) and associate with Sertoli cells to form primitive testicular cords (Brennan et al., 2004). Within the testicular cords, the primitive germline stem cells (now termed gonocytes) remain in the testis as the gonad differentiates. Eventually, Sertoli cells, peritubular myoid cells and gonocytes form more compact structures known as the seminiferous tubules. When the gonocytes migrate to the periphery of the tubules, they transform into prospermatogonia and then into spermatogonia (Gondos & Hobel, 1971). In the fetal testis, prospermatogonia enter mitotic arrest, a feature observed at E12.5 to E14.5 in the mouse. At the molecular level, during entry into meiosis, both male and female human gonocytes express DAZL proteins and Vasa transcripts and downregulate OCT3/4 expression (Anderson et al., 2007). Interestingly, it is the early migrating germ cells that share similar properties with embryonic stem cells and testicular germ cell tumors (Ezeh et al., 2005). From what is known, the development of gonocytes in the fetal ovary follows a similar path in that OCT3/4 expression is reduced while VASA, Germ Cell Nuclear Antigen (GCNA) and DAZL are expressed (McLaren, 2003). In contrast to the male gonocytes, the female gonocytes receive sex-specific signals from the fetal gonad to enter meiotic prophase. After initiating meiosis, the female gonocytes will develop into primordial follicles and subsequently into primary follicles at puberty. A key difference between mouse and human systems is the timing of primary follicle formation: the mouse achieves this stage at birth while in the human ovary, this occurs at puberty (Bukovsky et al., 2005). There is some speculation whether this difference in follicle development is due to autocrine signals produced from the oocyte itself or from the ovarian environment (Hutt & Albertini, 2007). A plausible hypothesis is that the immediate environment of early germ cells determines whether they are commit to spermatogenesis or oogenesis. The most obvious source of signals are the mesonephros, primitive Sertoli cells in the testis, and primitive Granulosa cells in the ovary.

Timeline of Germline Specification and Germ Cell Marker Expression. A temporal representation of the stages of human and mouse germline differentiation in vivo. At each cellular stage, important molecular and somatic signals controlling that stage are indicated above the diagram. Specific germ cell molecular markers are indicated on left with arrows depicting the duration of development during which they expression has been observed. Genes that are italicized are present in both mouse and human germ cells. At the bottom, an approximate timing of each stage during mouse or human germline development is indicated. Adapted from Schuh-Huerta & Reijo Pera, 2011.

The seminiferous tubule serves as the sperm production center, where approximately 123 x 106 spermatozoa are produced from germ cells daily, or about 1000 sperm/second (Amann et al., 1980; de Rooij, 2009). Developing germ cells are arranged along the basement membrane in a highly ordered sequence and extend into the lumen of the tubule. At the most basal portion of the tubules lies the spermatogonial stem cell (SSC) population, closely associated with the adjacent Sertoli cells. Morphologic analysis of the various germ cells reveals at least 13 recognizable germ cell types in the human testis (Heller & Clermont, 1963, 1964). Each cell type is thought to represent a different step in spermatogenesis. From the least to the most differentiated, they have been named dark type A spermatogonia (Ad); pale type A spermatogonia (Ap); type B spermatogonia (B); preleptotene (R), leptotene (L), zygotene (z) and pachytene primary spermatocytes (p); secondary spermatocytes (II); and Sa, Sb, Sc, Sd1, and Sd2 spermatids (Figure 3). The early, type A spermatogonia are the most interesting germ cell type from a stem cell point of view (de Rooij, 2009). In fact, time-course studies using GFP-based reporters with early type A, type Ad and type Ap spermatogonia in the mouse revealed that the early type A cell has the ability to divide, self-renew, and give rise to the Ad and Ap sun-populations (Nakagawa T. et al., 2007). These observations provide evidence for the existence of SSCs in the testis and their clonal behavior is protypical of other adult stem cells.

It is currently thought that pale type A (Ap) spermatogonia in the basal, stem cell niche of the seminiferous tubule divide at 16-day intervals and differentiate to type B spermatogonia, which then become spermatocytes (Clermont, 1972). The ability of SSCs within the testis stem cell niche to undergo stem cell renewal is governed by several known factors. The growth factor-receptor kit ligand/c-kit receptor system and the niche factor glial cell line-derived neurotrophic factor (GDNF) are important in this process (Oatley & Brinster, 2008). In fact, spermatogenesis in the rat is dependent on c-Kit receptor activity, whereas spermatogonial stem cell renewal may be c-kit independent (Dym, 1994). GDNF appears to provide a significant stimulus to self-renewal of SSCs through receptors for GDNF on SSCs such as c-Ret and GFR-1(Meng et al., 2000). Despite this, our knowledge of other receptor-ligand systems that control human SSC renewal is limited at this point. During spermatogenesis, the cytoplasm between spermatogonial daughter cells remains conjoined after mitosis, forming cytoplasmic bridges between adjacent cells (Ewing et al., 1980). Cytoplasmic bridges are thought to be important for synchronized cellular proliferation, differentiation, and possibly regulation of gene expression. Thus, SSCs and early spermatogonia in the adult testis are critical for germ cell renewal and differentiation into sperm and raise important questions about the source of proliferative and differentiation signals for spermatogenesis. The majority of stages in mouse spermatogenesis delineated above are translatable to the human germ cell development pathway except for differences in the timing of each stage during development.

Continue reading here:
Embryonic Stem Cells and the Germ Cell Lineage | InTechOpen