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1. Introduction
The gametes are highly specialized haploid cells that harbor genetic background of each individual. During fertilization, the fusion of female and male gametes occurs in order to produce zygote. These zygotes are diploid cells and have genetic material of both individuals. Zygotes start to divide and undergo further pre-implantation development through the formation of morula, blastocysts and finally fetus (Fig. 1). When intrauterine fetal development is finished, the organism is already formed and ready to birth. The zygotes can be considered primordial stem cells, which originate the whole organism through unequal divisions to produce blastomeres, the cells resulting by cleavage of a zygote. Sixteen blastomeres constitute a morula, the spherical embryonic mass surrounded by the zona pellucid, which further became a blastocyst. Blastocyst is a thin-walled hollow structure surrounded by trophoblasts layer that contains a cluster of cells called the inner cell mass (ICM) from which the embryo arises and the scientists isolate embryonic stem (ES) cells for
Figure 1.
Early development of stem cells niches. According to current knowledge there are natural stem cells niches during development: morula, blastocyst, epiblast and fetus, and artificial stem cells niches: stem cell culture
2. Natural in vivo pluripotent stem cell niches
2.1. Morula stem cell niche
Here we proposed that starting from morula, when zona pellucid arises, a first specific compartment called stem cell niche is formed. This niche can be defined as a microenvironment in which stem cells are found. Stem cell niche provides the both interaction between the cells and their interaction with local microenvironment, by which their fate regulates and occurs. In morula, stem cell niche consists of pluripotent stem cells that provide expression of specific transcription factor, such as POU domain transcription factor (Oct3/4) responsible for self-renewal capacity and pluripotency of these cells. In mammals morula first cell fate decisions is governed by key transcriptional factor: Oct3/4 (Palmieri et al., 1994). Oct3/4 is unique because it requires maintaining the pluripotency in both conditions
2.2. Blastocyst stem cell niche
The first lineage segregation is resulted in the formation of trophectoderm and ICM (Wobus, et al., 2005). Upon silencing of Oct3/4, a part of morula cells spontaneously inactivates the self-renewal process and start to differentiate into trophoblast cells, thus forming pluripotent stem cell niche in blastocyst. This blastocyst niche is a dynamic structure which follows developmental program of an organism in parallel with Oct3/4, expression of other transcriptional factor such as Nanog occurs in ICM. in early blastocyst (Nichols et al., 1998; Avilon et al., 2003).
2.3. Epiblast stem cell niche: Naive and primed pluripotent stem cells
In mice in late blastocyst transcription factor Sox2 starts to express in the cells of ICM in addition to GATA6 and Nanog, which lead to formation of two distinct populations: epiblast and hypoblast (Mitsui et al., 2003). These cell populations are considered the precursors of the primitive endoderm and the pluripotent epiblast (Morrissey et al., 1998). Recent studies suggest that stem cells in rodent epiblast have two distinct stable states of pluripotency: naïve and primed, thus establishing epiblast stem cell niche (Tesar et al., 2007; Nichols, 2009). According to these classification both of states exhibit features of bona fide pluripotent stem cells, such as have indefinite self-renewal, tri-germ layer potential and depend on expression of all three transcription factors, such as Oct3/4, Sox2 and Nanog (Tesar et al., 2009; Nichols, 2009; de Los Angeles et al., 2012). Naïve (more immature) pluripotent stem cells can be obtained from pre-implanted stage of embryo in rodents (Okamoto et al., 2003). These cells have both sex X chromosomes activated and are able to produce high-grade chimeras after their reintroduction into the host blastocyst. In contrast, in humans primed pluripotent ES cells are isolated from human pre-implantation blastocysts stage of development. In these cells one of female X chromosome is inactivated, albeit human ES cells are self-renewing and express key transcription factors and are able to form teratoma (Okamoto et al., 2003; Brons et al., 2007; Tesar et al., 2005). Studies of X chromosome inactivation in pre-implantation human embryos reported that
3. In vitro pluripotent stem cell niches
After isolation, pluripotent stem cells start to organize
4. Generation of artificial pluripotent stem cell – Reprogramming strategies
4.1. Reprogramming by means of differentiated cells nuclear transfer
Several strategies can be provided in order to reprogramming differentiated or committed somatic cell genome. One of these strategies is a nuclear transfer (NT) of differentiated cell nucleus to oocyte whose maternal DNA was removed (Campbell et al., 1996). This type of reprogramming uses the natural components without any previous genetic or molecular modification of nucleus–donor and oocyte-recipient. NT is relatively efficient and frequently depends on technical experience of researcher (Galli et al., 2012). There are two kinds of nuclear transfer trial: egg-NT involves the transfer of a single somatic nucleus to an unfertilized enucleated oocyte and oocyte-NT involves the transplantation of multiple somatic cell nuclei into immature oocyte of amphibian. Nevertheless are important differences between the two types of nuclear transfer experiment. In oocyte-NT experiments extensive cell division take places and new functional cell types appear as soon as the nuclear transplant embryo start to develop. In this experiment somatic cell chromatin is directly reprogrammed to express pluripotency genes within a day. In contrast to oocyte-NT experiments, in egg-NT no new cell types are formed, and neither oocyte nor nuclei divide, however direct transition of reprogrammed nuclei that transcribe genes of pluripotency into differentiated cells occurs. Analysis of the mechanism of reprogramming in egg-NT experiments, which involves transcription pluripotency genes and others, is complicated owing to rapid DNA replication and numerous cell divisions (Halley–Scott et al., 2010; Julien et al., 2010).
The NT process leads to direct reprogramming of pluripotent stem cell and expression of such markers as Oct3/4, Nanog, and Sox2 that are silent in differentiated somatic cell nucleus. In general, the reactivation of silent pluripotency genes starts around 24 and 48 hours after NT (Halley–Scott et al., 2010; Julien et al., 2010; Byrne et al., 2003). Upon NT occurs the series of events when oocyte cytoplasm induces changes in the structure of donor chromatin toward pluripotent state, which became more appropriate for embryonic development. However, synchronization process which should happen between genomic DNA of donor cell and cytoplasm of recipient cell is complex and may affect significantly pluripotency of reprogrammed cells. Attempts to facilitate this reprogramming process have been made using chemicals that alter the methylation status of the chromatin, such as TSA (trichostatin A), azacytidine, scriptaid, either before or after NT. In the mouse, the use of TSA (a histone deacetylase inhibitor, HDACi) significantly increased the success rate of mouse cloning (Kishigami et al., 2007).
Figure 2.
Stages of nuclear transfer. The nucleus is removed from an egg (or oocyte) and replaced by a nucleus from a donor cell (somatic cell).
In mammals, embryo obtained by NT and transferred into foster mother can result (or not) in full term development. The clones, obtained by NT method, are genetically identical to donor organism, which provide a nucleus. The sheep Dolly was the first successfully cloned farm animal. Dolly was obtained from NT of terminally differentiated mammary epithelial cell (Campbell et al., 1996). However the generation of animals by NT is not very efficient, once many clones are dying soon after implantation, and only few clones survive and born (Galli et al. 1999; Ritchie 2006). These clones frequently affected with severe abnormalities, they die prematurely and often obese. The survival rate of clones depends on species, on donor cell type, method of NT and varied significantly between different laboratories (Oback & Wells, 2002; Wilmut et al., 2002). However, pre-implantation development does not seem to be a problem (Ono et al., 2001; Ono et al., 2001a) the majority of the term losses occurs during the post implantation period and/or after birth. It has been reported in some experimental studies, that only 2-3% of the transferred embryos develop to term in mice (Ono et al., 2001a; Sakai et al., 2005). Over time the methods were improved and other species have been cloned with success from differentiated donor cells, such as cattle (Galli et al., 1999); mouse (Wakayama & Yanagimachi, 1999); pig (Polejaeva et al., 2000a); cat (Shin et al., 2002); goat (Keefer et al., 2002); mule (Woods et al., 2003); horse (Galli et al., 1999); rabbit (Challah-Jacques et al., 2003); rat (Zhou et al., 2003) and dog (Lee et al., 2005). In humans, the attempt to NT has been achieved using animal oocytes as recipients for human genetic material. The reprogramming of human somatic cell nuclei did not occur after NT into bovine and rabbit oocytes. These oocytes with human genome were not able to follow early embryonic development. The up-regulation of human pluripotency-associated genes did not occur. These data raised a question about the potential use of animal embryonic environment to generate patient-specific stem cells using NT technology. Ethical implications also should be taken in consideration (Chung et al., 2009).
4.2. Reprogramming by means of stem cells nuclear transfer
In 1998, Cibelli performed stem cells nuclear transfer (SCNT) using nucleus of bovine fibroblasts and enucleated bovine oocytes. They obtained 330 reconstructed oocytes, generated 37 cloned blastocysts, which served for isolation of 22 ES-like cell lines. These ES-like cells were injected into bovine oocytes, cultured cultured to produce embryos that further which were transferred into recipient females. In six out of seven calves at least one tissue originated from ES cell has been found. Other authors demonstrated the ability of karyoplast of ES cells induce Oct4 expression in the somatic genome (Tada, 2001).
In humans (Hall et al., 2007) and non-human primate (Mitalipov et al., 2002) the SCNT efficiency of blastocyst formation has typically been very low, thus suggesting a lack in or complete nuclear reprogramming. In order to overcome these difficulties modified SCNT approach was used to produce rhesus macaque blastocysts from adult skin fibroblasts and to isolate from this blastocyst two ES cell lines. This was achieved thought non-invasive approaches for meiotic spindle detection in oocytes and their removal using high-performance imaging. Spindle imaging system supports rapid and highly efficient real-time enucleation of primate oocytes. In this experiment spindle removal efficiency was 100%. The investigation of karyotype, microsatellite and single nucleotide polymorphisms (SNP) analyses confirmed that both ES cell lines were originated from SCNT embryos and were not from parthenotes. These ES cell lines demonstrated typical pluripotent cells morphology, self-renewal capacity and expression of stem cell markers. They were also transcriptionally similar to ES cells derived from fertilized blastocysts, and pluripotent, as demonstrated by the generation of several tissues from three germ layers after
The main goal of NT technology was to multiply the genotypes of high genetic value in farm animals and species, which are under the risk of extinction. Further, this technology was used as a tool for genome reprogramming of somatic differentiated cells into pluripotent state. The principles of cloning, which were developed by Willadsen (1986), are also important today. All the cloning studies provided the first experimental evidence for reprogramming (Kono et al., 1997; Gurdon, 2008). Currently, NT technologies can be applied in two different ways, to produce animal clones and to reprogram the nuclei of differentiated somatic cell, which can be used for basic research to analyze X chromosome inactivation or to study the dynamics of imprinting process during reprogramming and in some cases for pre-clinical evaluation of these cells in animal models (Hochedlinger and Jaenisch 2006). This technology yet holds medical interest to produce patient-specific stem cells, which can be used in cell therapy and regenerative medicine.
4.3. Reprogramming by means of early embryonic environment
The pluripotency, characteristic feature of ES cells, can be evaluated by their capacity to differentiate into cells of the three germ layers. More precisely, ES cells pluripotency can be evaluated by generation of chimaeras, organisms composed of cells from two or more individuals from the same or different species (Kaufman, 1981; Keller, 1995; Wobus, 2005). Production of human/animal chimaeras is a method currently in use to analyze developmental potency of mammalian ES in biomedical research (Behringer, 2007; Lensch et al., 2007). James et al (2006) showed for the first time that a nonhuman embryo surrogate environment could be used to study developmental potential of human ES cells as well as biological compatibility between human ES cells and the mouse ICM. Adult stem cells (ASC) are now seen as an alternative to ES cells, which can raise a number of ethical objections due requires destruction of human embryo. Populations of multipotent ASC that express ES cell markers, such as Oct3/4, Nanog and Sox2, presenting a differentiation capacity similar to that of ES cells
Figure 3.
The hIDPSC injection in early embryonic environment. (A) hIDPSC stained with vital Vibrant fluorescent dye were injected into the perivitelline space and/or compacted morulae (B) hIDPSC showed a contribution to the ICM and also to the trophoblast cell layer.
Additionally to Vibrant fluorescent dye the anti-hIDPSC antibody, (this identifies exclusively hIDPSC), was used to detect the presence of these cells in 18 d.p.c. mouse foetuses). Strong fluorescent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine and muscles (Fig. 4D and Fig. 5B). Using a variety of methods we demonstrated hIDPSC contribution to mouse embryos, which did not present any type of morphological deficiency (Fig. 5A). We were able to produce evidence, that these cells accomplished differentiation within local tissues, by the presence of human-specific tissue proteins, such as myosin and cytokeratin. Moreover, we used a specific antibody against human nuclei to confirm, again, that the cells were indeed of human origin (Siqueira da Fonseca et al., 2009). Little is known about the initial reprogramming events that occur after transference of ASC into mouse blastocysts (Yokoo et al., 2005). In our experiment, hIDPSC were capable of engrafting and proliferating inside mouse morulae and blastocysts and forming pretermed chimaeras. These cells contributed not only to ICM, as do human ES cells, but also to the trophoblast cell layer – without any embryo damage.
Figure 4.
Developmental and pluripotent capacity of hIDPSC to generating pretermed human/mouse chimaera. Early chimera blastocyst (A) were transferred to the uterus of foster mother (B). Human/mouse chimaeras (C) were collected before birth and fluorescent signals were observed in different organs of the chimaeras (D).
Furthermore, hIDPSC integrated into host embryos and developed foetuses, undergoing the process of differentiation. Obviously that due to the difference in cell cycle dynamics between mouse and human cells, the number of human cells during mouse pre-natal development is decreased in comparison with hIDPSC contribution in ICM of blastocyst. However, it is not clear if hIDPSC can really undergo reprogramming into ES-like cells within nonhuman embryo surrogate environment.
Our finding suggests that expression of such pluripotent markers, as nanog and oct4 by hIDPSC is enough condition for these cells to contribute into different mouse tissues in early embryo-fetal development, to differentiate properly and to express human proteins within mouse fetal an immune privileged environment (Siqueira da Fonseca et al., 2009).
Figure 5.
The hIDPSC contribution in pretermed human/mouse chimaera. (A) 18 d.p.c. mouse fetus. (B) Strong fluorescent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine, muscles and others.
4.4. Reprogramming by means of cell fusion
First pluripotent hybrid cells have been isolated by fusion of pluripotent teratocarcinoma (TC) cells with differentiated somatic cells, which served as a tool for investigating the interaction between different genomes. These TC cells are similar to ES cells in morphology and gene expression pattern, thus maintaining variable levels of pluripotency, however not all TC cells able to generate chimaeras and to contribute to germ line (Papaioannou and Rossant 1983). These cells frequently have abnormal karyotype, such as loss of the Y chromosome, trisomy, deletions or translocations (Takagi et al. 1983, Rousset et al. 1983, Modlinski et al. 1990). The hybrid cells obtained from pluripotent TC cells and somatic cell partner, which express embryonic antigens, were able to produce teratomas containing derivatives of all three embryonic germ layers (Andrews and Goodfellow 1980, Atsumi et al., 1982; Rousset et al. 1983; Forejt et al., 1984; Takagi, 1983) and to form embryoid bodies (EBs) in suspension culture (Takagi, 1983). These hybrid cells showed also reactivation of particular genes after reprogramming (Miller and Ruddle, 1976, 1977; Andrews and Goodfellow, 1980; Rousset et al., 1983) and reactivation of inactive X chromosome originated from the somatic partner (McBurney and Adamson, 1976; McBurney and Strutt, 1980; Takagi et al., 1983, Takagi, 1988; Mise et al., 1996). However, pluripotent hybrids were obtained when lymphocytes or thymocytes, not fibroblasts, were used as the somatic parents in fusion (Rousset et al., 1979). These studies indicate that hybrid cells generated by ES cells and differentiated cells, which have less cytoplasm, seem to be more adequate systems to undergo reprogramming.
Matveeva et al. (1996) has obtained cultures of intraspecific embryonic hybrid cells by fusion of mouse ES cells, denominated HM-1 cells, which were derived from HPRT-deficient strain 129 mice (Magin et al., 1992) and characterized as highly pluripotent (Magin et al., 1992; Selfridge et al.1992) with splenocytes derived from an adult DD/c female. These hybrids were denominated as hybrid embryonic stem and somatic (HESS) cells and characterized as pluripotent and HPRT positive (Matveeva et al., 1996; 1998). Our group used three mouse hybrid clones HESS-1, HESS-2 and HESS-3 in order to study their karyotypes and investigate the influence of the karyotypes on the differentiation of these cells through the formation of embryonic bodies (Mittmann et al., 2002). The hybrid cells used in our study were near diploid (HESS-2 and HESS-3) and near tetraploid (HESS-1) and chromosome analysis showed different trisomies. The trisomies of chromosomes 1 and 11 were found in near diploid hybrids. These trisomies are probably typical of these pluripotent cells, and have previously been described in the mouse ES cells line (Crolla et al., 1990) and in TC cells (McBurney and Rogers, 1982). We found that the sex chromosome constitution in the HESS-2 line was predominantly XY, while in the HESS-3 line it was XO. Interesting that in HESS-2 and HESS-3 lines the segregated X chromosome was of embryonic origin. Indeed, it has been demonstrated by Ringertz and Savage (1976) that hybrids lose the chromosomes originating from differentiated, more slowly dividing cells. In our experiments, hybrids showed the capacity to form EBs
In the EBs derived from hybrids we observed haematopoietic-like cells, cells resembling skeletal and smooth muscle and others (Fig. 7). Cells of ectodermal origin (e.g. nerve cells) were not identified in EBs derived from hybrids. Our data shows that the ‘embryonic’ X chromosome may be lost in pluripotent hybrids, but reprogramming of the ‘somatic’ X chromosome may still occur, thus allowing restricted pluripotency. The normal karyotype may be a prerequisite for the efficient contribution of these cells to the germ line in transgenic and chimeric animals and for their ability to differentiate
Figure 6.
Cystic embryoid body - a globular cell cluster cultured from mouse ES cells.
Therefore, we demonstrated that near diploid somatic cell hybrids obtained by the fusion of ES cells with differentiated cell can be fully reprogrammed and able to produce
Figure 7.
Electron microscopy demonstrates differentiation within cystic embryoid bodies derived from somatic cell hybrids (HESS-2).
Figure 8.
Germ cell derived in vitro from HESS-2. (A) Protocol of differentiation. (B) Expression of genes involved in the progression of early stages of gametogenesis during HESS-2 differentiation. (C) Sperm-like structure. (D) Oocyte-like structure. (E and F) Haploid cells with X or Y chromosomes. (G) Expression of oocyte-specific markers in oocyte-like structures obtained from HESS-2.
4.5. Reprogramming by means of Yamanaka’s factors
The pluripotency manifests during short time of early mammalian development (Choen et al., 2011; Dejosez et al., 2012). Such powerful, pluripotent cells can be obtained
Reprogrammed hIDPSC presented all key characteristics of pluripotent cells: formed juxtaposed colonies of ES-like morphology and produce teratoma with derivates of all three germ layers. These cells did not integrate retroviral vector in their genome and express lower levels of Oct4, Nanog and Sox 2. In contrast to iPS cells derived to fibroblast cell, the hIDPSC derived iPS cells were generated to in shorter time and presented higher efficiency of colonies formation And were able to form under iPS colonies feeder –free conditions conditions. For example, the time of fibroblasts reprogramming using retrovirus vectors takes 20–25 days (Aesen et al., 2008), while reprogramming of hIDPSC occurs only in eleven days after infection (Beltrão-Braga, 2011). These results suggest that age of donor and differentiation status of cell type used for reprograming may also affect reprogramming efficiency. Accordingly, Maherali and Hochedlinger et al., (2007) compared skin fibroblasts reprograming efficiency from two-month-old and two years-old mice. Older cells produced half as many iPS cell colonies as young skin fibroblasts. It has been shown that iPS cells have so-called epigenetic memory, which means that after reprograming their differentiation potential can reflect on their lineage commitment before reprograming. Therefore, hIDPSC showed strong neural commitment, which is due to their ectomesodermal origin. After reprograming strong neural commitment was evidenced within teratomas as well as spontaneous
Figure 9.
hIDPSC-derived iPS cell. (A) Representative figure of morphological characteristics of hIDPSC
5. Final considerations
All reprogramming strategies are aimed at genomic reprogramming, which is a key biological process. It is still unknown, how many and what reprogramming factors, which initiate a cascade of reprogramming events, are involved in NT, SCNT, in cell fusion and even in iPS cell production. Yamanaka’s study suggests that these factors may be mainly proteins of the nucleus; however the cytoplasm factors also should be taken in consideration. NT technique, which was used for Dolly the Sheep and many other species, has been abandon by many researches due to the low efficiency. Some researches try to use SCNT in stem cells research in order to obtain stem cells that are genetically matched to the donor organism. However, up to data no human ES cells were obtained using SCNT. Another limitation of this method is that resulting cells retain mitochondrial structures, which originally belonged to the egg. The great limitation of cell fusion technology is chromosome set composed by different genomes. Currently many scientists, which used all these methods moved to iPS cell production.
We started this chapter with simplified description of the concept of stem cell niches formation during early development. This conception lead to comprehension that such niches are very complex and composed by heterogeneous population of different somatic and stem cells. We know, that at least two different populations of pluripotent stem cells naïve and prime can be identified
Acknowledgments
The authors thank Dr. Thais M. C. Lavagnolli from Imperial College London and Dr. Simone A. S. da Fonseca from University of Sao Paulo for their contribution in our research.References
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