Embryonic Stem Cells and creating artificial gamets and embryo’s.

The number of infertile men and woman has increased from 10 to 15% in the past 11 years. The fact that these number are increasing means that we need to progress the field of cloning artificial gamets and embryos in reproductive medicine. However, current scientific advancement cannot offer help for patients who are completely sterile. They can not produce healthy embryos of their own but want their own offspring. One of the reason these kind of patients can not get own offspring I because of, ovarian cancer and chemoradio therapy of cancer in men are common causes.

If assisted reproductions fails the only other option is to use donor embryos, patients without functional embryos, and homosexual couples who really want own offspring can and in some countries do choose this path. To get this technology work they need Sperm cells but for cancer patients this is not bailable option. Since they lack sperms some patients undergo cryopreservation of testicular tissues which contain germ cells and apply it in fertility preservation strategy (Goossens et al., 2013; Picton et al., 2015; Gassei and Orwig, 2016).

These Germ cells obtained from testicular tissues and in vitro maturation can be used to fertilize oocytes and achieve pregnancy through Assisted reproduction technologies (Brinster and Zimmermann, 1994; Schlatt et al., 2002; Kim, 2006; Kim et al., 2008; Hermann et al., 2012; Saiz and Plusa, 2013). 

However, these kind of technologies need embryo research and ethical restrictions are the main obstruction for human embryo studies. Protocols for in vitro human embryo culture beyond the blastocyst stage need more optimization, and research beyond day 14 is prohibited which makes it even harder do research during and beyond   the onset of primitive streak (PS) developmen (Deglincerti et al., 2016; Shahbazi et al., 2016) (Daley et al., 2016; Hyun et al., 2016). Therefore, more research is required in reproductive medicine, and in cloning synthetic embryos in assisted reproduction. 

Assisted reproduction synthetic  embryos can be defined as embryos generated by manipulation and cloning of progenitor cells or somatic cells and stem cells to derive embryos assemble to their natural state, which provides a new possible therapy. The generation of assisted reproduction synthetic  embryos will provide therapeutic advantages clinically but also generate a terrific platform for studying developmental biology. Developmental studies on human germ cells and embryos are mostly based on animal models due to the lack of available human samples. Eventhough researchers have collected a lot of data about embryo formation but embryo development are species-specific, Which means that animal models can not easily be implemented on humans (Irie et al., 2015; Sugawa et al., 2015). 

The main goal in assisted reproduction synthetic embryos is the possibility of establishing a method so that ethical issues can be avoided. This will open the door for cellular and molecular research during the developmental process and establishing personal disease models.

Creating and cloning germ cells in vitro

The key goal of assisted reproduction synthetic embryos is to produce a functional egg or sperm by reconstituting the process of gametogenesis in culture. Germ cells are the precursors of sperm and egg cells, which generate the totipotent state.  germ cells arise from the proximal epiblast, which is a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac (Lawson and Hage, 1994); 

In general two kinds of stem cells can be used as material for generating  germ cells: adult stem cells from male and female gonads and pluripotent stem cells, which include embryonic stem cells (Aguilar-Gallardo et al., 2010) and induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007). 

The reconstitution of  germ cells has been explored in primates. Embryonic stem cells and iPSCs from cynomolgus monkeys (Macaca fascicularis, referred to as “cy”) are efficiently induced to differentiate into  germ cells bearing a transcriptome similar to that of early cy germ cells (Sakai et al., 2019). In humans, the specification of  germ cells has not been achieved. 

Embryos From Adult Stem Cells

Embryos from adult stem cells require expansion and directional differentiation. Adult stem cells have specific pluripotency; these cells can self-renewal and differentiation into limited cell types. Adult stem cells rely on the niche in vivo. The stem cell niche offers a specific microenvironment containing different metabolic factors, molecular pathway factors, sex steroids, immunologic protection, nutrition and even topology.

Why is it hard to culture germ stem cells

The function and fate of Spermatogonial stem cells are regulated by the niche, which refers to the microenvironment surrounding Spermatogonial stem cells that is mostly constituted by Sertoli cells (SCs). Which means that developing Spermatogonial stem cells into germ cells. This evidence has been supported by in vitro experiences. Although spermatogenesis has not been fully achieved in vitro, the most successful attempts to this point have been based on co-culture of germ cells with SCs (Griswold, 1998; Nagano et al., 1998; Zanganeh et al., 2013; Rebourcet et al., 2014; Xie et al., 2015; França et al., 2016). Sertoli cells secrete factors that direct germ cell fate.

In vitro experiments with Spermatogonial stem cells from non-human primates and humans cannot reach the standard Spermatogonial stem cells reached, which means establishing a testicular niche in vitro may require not only substances secreted by somatic cells but also the spatial structure. In addition, the number of somatic cells could be a large factor in the in vitro culture system because of their responsiveness to media additives and because they have shorter proliferation cycles (Gat et al., 2017). Spermatogonial stem cellss are mostly quiembryonic stem cellent. Hereafter, effectively controlling the number of somatic cells in the culture system may trigger the Spermatogonial stem cellss to proliferate.

For women, the presence of oogonial stem cells in postnatal mammalian ovaries is controversial, as it has long been held that the ovaries contain a fixed number of germ cells throughout a woman’s lifetime (Zuckerman, 1951). However, recent studies have provided evidence of mitotically active Oogonial stem cells in adult human ovaries (Johnson et al., 2004; Zou et al., 2009; Passisted reproduction technologiese et al., 2011; White et al., 2012). Which means that even after the menopause are still a source of oocytes that can be exploited to achieve fertility in women who are infertile or have an exhausted ovarian reserve, as long as the genetic integrity of the Oogonial stem cells is maintained (Virant-Klun et al., 2008; Woods and Tilly, 2012; Dunlop et al., 2013; Gheorghisan-Galateanu et al., 2014).

Even though the existence of Oogonial stem cells is in dispute, there have been studies that generated oocytes or oocyte-like cells from Oogonial stem cells. Similar to Spermatogonial stem cellss with Sertoli cells, somatic cells also play an important supporting role in oocyte formation. Oocyte-granulosa cell complexes are regarded as the stem cell niche in the female reproductive system. 

If Oogonial stem cells can be cloned expanded and differentiated into oocytes, this technique can be applied clinically for women who lack oocytes. However, there are still obstacles to overcome before these methods can be applied in the clinic due to the related ethical problems. In addition, whether assisted reproduction synthetic embryos are healthy enough to produce offspring is still uncertain.

Embryos From Embryonic Stem Cells

Embryonic stem cells have always been a good platform for studying in vitro cell lineage differentiation (Nishikaw

a et al., 1998; Rathjen et al., 1998). People lacking adult stem cells, the only remaining option is the transformation of patient-specific somatic cells into pluripotent stem cells, which is then followed by differentiation into genetically related haploid embryos. 

 

There are two ways to generate patient-specific pluripotent stem cells. First, the nucleus of a somatic cell can be transferred into an enucleated oocyte, which is also known as somatic cell nuclear transfer (SCNT) (Tachibana et al., 2013). This oocyte will develop into an embryo at the blastocyst stage, and the ICM can be retrieved. With these cells, a patient-specific Embryonic stem cell line can be generated. Second, somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007). In general, the iPSC is more commonly used in the initial steps of this process. It is believed that somatic cells constitute an important support system guiding differentiation, but most studies focus on either female or male in vitro gametogenesis.

Male haploid germ cells at the round spermatid stage have been derived from Embryonic stem cells by spontaneous differentiation in vitro. This offers the possibility of investigating germ cell development, epigenetic reprogramming, and germline gene modification. Embryonic stem cell-derived  germ cells can differentiate into sperm, as shown by transplantation experiments wherein the form MVH-expressing spermatogenic cells. Injecting Embryonic stem cell-derived  germ cells into oocytes can restore the somatic diploid chromosome complement and can enable development into blastocysts (Toyooka et al., 2003; Geijsen et al., 2004). For human in vitro reconstitution of oogenesis we can tell that the culturing system of mouse oogenesis is fully developed, but a similar system cannot be replicated in humans. Obstacles exist, including the complicated events that occur during oogenesis.

Embryos made from Stem Cells

Assisted reproduction synthetic embryos might be a better solution to bring offspring. The goal of assisted reproduction synthetic embryos is to establish embryo-like structures without any germ cells; some researchers have focused on the differentiation of Embryonic stem cells to make these structure, while others have found that assembling several types of stem cells might be feasible. 

Many studies have focused on generating blastocyst-like structures by aggregating several kinds of stem cells. Rivron et al. aggregated mouse embryo stem (ES) cells for 24 h and covered these non-adherent aggregates with trophoblast stem (TS) cells to form blastoids that were similar to E3.5 mouse embryos; the formation frequency was low (0.3%). 

The construction of embryo-like structures in vitro can offer a model for studying fundamental biological questions in both preimplantation and early post-implantation mammalian embryogenesis and can enable the modeling of diseases related to early pregnancy, the performing of high-throughput pharmacological and toxicological screens, and possibly the bioengineering of mammalian embryos. The development of early mammalian embryos is plastic and is regulated by several evolutionarily conserved developmental processes that can be recapitulated in vitro. Assisted reproduction synthetic embryos are desired not only in mice but also in other mammalian species, including humans. However, the derived reorganized embryos exhibit features of different embryonic stages, but they are not equivalent to totipotent blastomeres. A deeper understanding of these differences is required to build a better in vitro environment for natural embryos. In addition, to date, no assisted reproduction synthetic embryos derived from the organization of stem cells develop normally, and fertile offspring have not been reported. This indicates that assisted reproduction synthetic and reorganized embryos remain a substantial challenge in the field, and more investment and research are required.

The Possible Uses of synthetic Embryos

Assisted reproduction synthetic embryos have scientific uses. Due to ethical restrictions, there are areas where little is known in human embryonic development. However assisted reproduction technologies from the research purpose, assisted reproduction synthetic embryos are expected to have clinical use; they might represent another available treatment for infertility. Instead of donated sperm or eggs, assisted reproduction synthetic embryos could bring hope for genetically related offspring. In addition, patients who lost their fertility because of cancer treatment, including pediatric cancer patients, might regain fertility. These tools also offers another choice of fertility preservation. The safety of assisted reproduction synthetic embryos is of special concern. Although fertile offspring can be derived in several rodent studies, these protocols cannot guarantee success in more advanced mammalian species, including humans. Assisted reproduction synthetic embryos will become a powerful research platform for early embryo development, especially human embryonic development.

Gametogenesis can be initiated from Embryonic stem cells or adult stem cells. The number of adult stem cells is very limited in both male and female gonads; therefore, cloning of adult stem cells is a critical step before gametogenesis. For Embryonic stem cells, the limited number of cells is no longer a concern, but it always takes multiple steps to accomplish differentiation. Whether important information has been fully maintained is uncertain, and whether deriving assisted reproduction synthetic embryos directly from the PSC stage is feasible and preferable is also unknown. The experience derived from mouse models can be inspiring for humans but cannot be translated directly. More experience is required on advanced mammalian species, such as nonhuman primates and humans. The significance of the stem cell niche in the testis and ovary is realized but not fully understood in humans; thus, reconstitution cannot be fully implemented. The field of assisted reproduction synthetic embryos has just assisted reproduction technologies, and additional work is required.

 


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