Mammalian Stem Cell Lines

Following the derivation and culture of human embryonic stem cell lines in the late 1990s (Thomson et al., 1998), debate and controversy escalated surrounding the use of surplus human embryos donated for research. Despite the controversial ethico-legal perspectives, in many countries throughout the world IVF clinics have embryos that are either unsuitable for treatment or are surplus to the patient’s requirements. Given the opportunity for appropriate counseling and informed consent, many patients choose to make a contribution to science by donating surplus embryos for research rather than allowing them to perish (Franklin et al., 2008). There is no doubt that embryos donated for stem cell research represent a very valuable resource for scientific investigation, with the potential to make a significant contribution to our understanding of early developmental processes and the molecular pathology of disease. Stem cell biology has become an integral part of ART; the principles and the science underpinning this new area of developmental and regenerative biology should be viewed within the perspective and frame of reference of the first stages of postimplantation development, as presented in the brief synopsis in Chapter 6.

Human Embryonic Stem Cells (hESCs)

Human embryonic stem cells are derived from the in-vitro expansion of epiblast progenitors within the inner cell mass of a preimplantation blastocyst. hESCs are capable of indefinite self-renewal, and maintain the potential to differentiate into cell types from the three embryonic germ layers (pluripotency). Because they remain pluripotent when maintained in vitro, they provide an ideal resource for investigating and studying the pathways that lead to the establishment of cells that might be relevant to clinical treatment, such as dopamine-producing neurons and insulin-producing cells of the pancreas.

The goal of hESC research is to elucidate the pathways that direct differentiation in vitro, with the aim of providing functional and therapeutically relevant cells. These cells can be used to investigate mechanisms of disease progression, and for research into drugs that might inhibit or reverse pathological processes. hESCs also provide an insight into aspects of early embryonic development that are otherwise inaccessible to research, due to ethical and practical considerations; they can be used as a tool to investigate how cells can be manipulated to regenerate damaged or diseased cells in the human body. Lastly, hESC-differentiated cells may eventually prove to be useful in cell transplantation approaches for the treatment of disease (see Trounson & DeWitt [2016] and Chen et al. [2014], for reviews).

Several strategies have been applied to promote the differentiation or selection of therapeutically relevant cell types, including the addition of growth factors or cytokines, as well as other ways of manipulating gene expression. One major hurdle to this approach is that the process of directing the differentiation of hESCs into functionally relevant specialized cells is very inefficient, and their intrinsic potential can allow them to switch from pluripotency toward uncontrolled differentiation during expansion in culture. This inefficiency may be the result of heterogeneity within a starting stem cell population, in addition to the lack of information about the developmental events that promote the emergence of specialized cells in vivo – differentiation into a particular lineage is a highly complex process, controlled by a multitude of overlapping parameters. The constraints of in-vitro culture conditions also hinder the ability to mimic in-vivo events.

The large-scale availability of treatments involving pluripotent stem cells will require further research to elucidate the fine details of the growth and stability of the cells, as well as a secure and thorough regulatory pathway to ensure their safety.

Tissue stem cells in fetal or adult tissue can supply cells to parts of the body that require a continuous supply of regenerative cells. Tissue stem cells include blood and intestinal stem cells; these are partially committed cells that can function to regenerate their respective adult tissues as required. They commonly reside in a microenvironment (niche) in a quiescent state where they are provided with signals and support that promote the maintenance of self-renewal. Stem cells exit the niche as they undergo cellular commitment/differentiation.

Multipotent Stem Cells

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  All blood cell types are continuously replaced from a store of hematopoietic stem cells (HSCs) in the bone marrow.

  
  
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  The lining of the gut (gut epithelium) has intestinal stem cells in the small intestine that produce four different cell lineages (Paneth, goblet, absorptive columnar, enteroendocrine).

  
  
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  Epidermal stem cells in the skin and hair follicle can regenerate damaged epithelium.

  
  
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  Skeletal muscle stem cells (satellite cells) are quiescent cells that can also give rise to committed progeny such as myofibers in response to injury or disease.

Oligopotent Stem Cells

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  Neural stem cells are restricted self-renewing subtypes that give rise to three lineages: neurons, oligodendrocytes, astrocytes.

Unipotent Stem Cells

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  Spermatogonial stem cells give rise to spermatogonia.

Differentiation

Cells can be distinguished from one another by their patterns of gene expression, which includes expression of both protein coding and noncoding RNAs, the secretion of proteins, their response to extracellular signals and the distribution of epigenetic chromatin modification. Changes to any or all of these processes can influence the differentiation of stem cells. For example, hESCs express the transcription factors OCT4, NANOG and SOX2 and require extracellular signals such as fibroblast growth factor (FGF) and Activin/Nodal to maintain self-renewal. Changes in the levels and balance between FGF and Activin/Nodal can influence the maintenance and differentiation of hESCs: in the presence of too much Activin/Nodal they resemble endoderm cells, and too little Activin/Nodal will cause differentiation into ectoderm cells. The mechanisms involved in the maintenance of hESCs is poorly elucidated, but is likely to involve cell signaling pathways that function at different levels, with multiple feedback controls and intercellular gene regulation:

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   1. Gene expression: transcription factors are proteins that function to promote or repress gene expression at the DNA level. For example, OCT4 is thought to maintain hESCs by binding to genomic regulatory regions to promote the expression of pluripotency-associated genes and repress the expression of genes associated with differentiation.

   
   
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   2. Extracellular signals: small proteins that are produced and secreted (cytokines) by a stem cell niche can contribute to the maintenance of self-renewal. These proteins are often required to maintain stem cells in culture, such as the addition of cytokines FGF and epidermal growth factor (EGF) to neural stem cells grown in vitro.

   
   
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   3. Chromatin modifications: DNA is packaged with histones to form nucleosomes. Post-translational modification of histone tails forms a code that can determine states of gene expression that are heritable, modifying gene expression by influencing the access of transcription factors to genomic regulatory regions.

   
   
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   4. Physical context: the presence of extracellular matrix and physical contact with other cells can influence the maintenance and differentiation potential of stem cells.

Early Culture Systems

During the early 1960s, Robert Edwards recognized the extraordinary potential of stem cell biology, and in collaboration with Robin Cole and John Paul in Glasgow, he isolated stem cells from the inner cell mass (ICM) of rabbit blastocysts. They cultured zona-free rabbit ICM on collagen surfaces, sometimes with HeLa feeder layers, and established cell colonies that showed differentiation to muscle, blood islands, neurons and complex groups of differentiating cells (Figure 7.2). Four immortal cell lines (two epithelioid and two fibroblastic) that survived more than 200 generations of subculture were established and cryopreserved. All cell lines remained diploid for several generations (Cole et al., 1965, 1966; Edwards, 2008).

(a) Cell masses derived from a zona-free rabbit blastocyst.

(b) Muscle differentiating after continued culture.

(c) A single blood island that developed among cell outgrowths from rabbit ICM.

(d) A group of neurons in the same outgrowths.

(e) A complex group of differentiating cells in which muscle cells are mixed with various types of differentiating cells – a pathologist discerned several cell types in this mass of cells.

Reprinted with permission from Edwards (2008).

Figure 7.2 Cell colonies derived from intact zona-free rabbit blastocysts after 20 days in culture (Cole et al., 1966).

In further studies by Richard Gardner (1968), ICM cells isolated from mice with marker (coat color) genes were injected directly into the blastocoelic cavity of recipient mice, forming chimeras which showed that the grafted cells had colonized several different tissues; this indicated that the injected ICM cells were multipotent. After human blastocysts first became available through IVF, Edwards and his team attempted to prepare human ESCs in vitro (Fishel et al., 1984), but this early work had to be abandoned due to ethico-legal problems surrounding the use of human embryos for research.

Embryonal Carcinoma (EC) and Embryonal Germ (EG) Cell Lines

In the early 1970s, stem cell lines that could be propagated in vitro were derived from murine teratocarcinomas (Kahn and Ephrussi, 1970); these cell lines were capable of unlimited self-renewal and multilineage differentiation. Mouse EC cells express antigens and proteins that are similar to cells present in the ICM, which led to the concept that EC cells are an in-vitro counterpart of the pluripotent cells present in the ICM (Martin, 1981); this provided the intellectual framework for working toward derivation of both mouse and human embryonic stem (ES) cells. Human EC lines were established in 1977 (Hogan et al., 1977), and these proved to differ from mouse EC lines, both in expression of surface markers, and in their in-vitro properties. Unlike mouse EC lines, the human cells are highly aneuploid and have a limited ability to differentiate into a wider range of somatic cell types. Nonetheless, these human cell lines provided a useful model in which to study cell differentiation (Andrews et al., 1984b; Thompson et al., 1984; Pera et al., 1989). Both mouse and human EC culture systems enabled numerous improvements in technique and methodology, as well as initiating studies on factors that are involved in the control of differentiation in vitro.

Stem cell lines derived from mouse testicular teratomas (EG cells) were found to contribute to a variety of tissues in chimeras, including germ cells (Stevens, 1967); this provided a practical way to introduce modifications to the mouse germline (Bradley et al., 1984). Pluripotent EG cells were successfully derived directly from primordial germ cells in vitro in 1992 (Matsui et al., 1992; Resnick et al., 1992).

Mouse Embryonic Stem Cells

Using culture conditions that had been used for mouse EC cells (inactivated fibroblast feeder layers and serum), the first mouse ES cell lines were derived from the ICM of mouse blastocysts (Evans and Kaufman, 1981; Martin, 1981). It was subsequently found that the efficiency of mouse ES cell derivation is strongly influenced by genetic background and that different culture conditions were required for strains that were initially thought to be nonpermissive. Further experiments revealed that mouse ES cells could be sustained by conditioned medium harvested from feeder layers, in the absence of the feeder cells themselves. Fractionation of conditioned medium led to the identification of leukemia inhibitory factor (LIF) as one of the cytokines that sustains mouse ES cells (Smith et al., 1988; Williams et al., 1988). LIF activates the transcription factor Stat3, which inhibits differentiation and promotes viability. Further investigation of the signaling pathways showed that the proliferative effect of LIF requires a finely tuned balance between positive and negative effectors/factors. If serum is removed from the medium, mouse ES cells can be maintained in an undifferentiated state by adding LIF in combination with bone morphogenetic protein (BMP), a member of the TGF-alpha superfamily (Ying et al., 2003).

The subsequent two decades of research using murine ESCs led to numerous advances in culture system techniques and technology, and the identification of several types of in-vitro differentiated cells (including neural tissue) introduced the therapeutic potential and hope of eventual therapies to treat degenerative diseases – neurodegenerative disease in particular. The murine ES model contributed enormously to many different aspects of developmental biology: a large collection of genes, factors, markers and signaling pathways involved in differentiation have now been identified, providing clues toward achieving directed differentiation of these pluripotent cells in vitro (Yu et al., 2007).

Human Embryonic Stem Cells

A considerable delay (17 years) ensued between the derivation of mouse ES cells in 1981 and the first establishment of human ES cell lines in 1998. This was due to at least two factors:

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   1. Suboptimal media and conditions for human embryo culture meant that human blastocysts were rarely available (especially for research purposes). Media optimized for extended culture were introduced during the mid-1990s, and blastocyst culture then became a more practical reality.

   
   
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   2. Initial culture systems for hESC derivation were based on those that were successful in the mouse, and it eventually became apparent that there are significant species-specific differences between mouse and human ES cells.

Isolation of ICMs from human blastocysts was reported in 1994 (Bongso et al., 1994), but they were cultured in conditions that allowed derivation of mouse ES cells, and this resulted only in differentiation of the human cells instead of their derivation into stable pluripotent cell lines. In the mid-1990s, ES cell lines were derived from two nonhuman primates: the rhesus monkey and the common marmoset (Thomson et al., 1995, 1996). Using experience gained from these primate models, in 1998 Thompson and his colleagues then reported the isolation of pluripotent stem cell lines derived from human blastocyst ICM cultured on inactivated mouse feeder layers. These cell lines expressed markers for pluripotency and could be maintained undifferentiated in long-term culture. The cultured cells also maintained the potential to form derivatives from all three germ layers when injected into severe combined immunodeficiency (SCID) mice. Media containing LIF and its related cytokines, required for mouse ESCs, failed to support human or nonhuman primate ES cells (Thomson et al., 1998; Dahéron et al., 2004; Humphrey et al., 2004); the fact that fibroblast feeder layers supported both mouse and human ES cells was apparently a fortunate coincidence.

Reubinoff et al. (2000) then reported directed differentiation of hESCs, producing three neural cell lines (astrocytes, dendrocytes, mature neurons) from an early neural progenitor stem cell in spontaneously differentiating cultures. A huge expansion in research activity followed this initial report, and over the next few years panels of markers associated with pluripotency and with stages of differentiation were identified. Research continues into elucidating pathways of differentiation and identifying factors that sustain hESCs in culture, as well as active factors that decay with de-differentiation, or change during re-differentiation. The body of published literature surrounding hESCs is now vast; by manipulating culture conditions, spontaneous differentiation to numerous different cell types has been observed, including beating heart cells (Mummery et al., 2002), insulin-secreting cells, hepatocytes, cartilage, etc. Protocols are now available for directing at least partial differentiation of hESCs toward numerous different fates: early endoderm, hepatic cells, pancreatic cells, cardiomyocytes, endothelial cells, osteogenic cells, hematopoietic cells, lymphocytes, myeloid cells, etc. (see Sullivan et al., 2007). hESC lines have also been genetically modified, with fluorescent reporter genes introduced into key gene loci that can be traced during in-vitro differentiation in order to identify subsets of cells along developmental pathways (Davis et al., 2008a; Hatzistavrou et al., 2009).

Figure 7.3 Transfer of a diploid somatic cell nucleus into an enucleated metaphase II oocyte followed by activation by electrofusion leads to the creation of an embryo that has the chromosome complement of the somatic cell. A resulting blastocyst stage embryo can be transferred to a recipient uterus and allowed to develop to term into an animal with characteristics of the transferred somatic nucleus (reproductive cloning), or inner cell mass cells may be used to generate stem cell lines that are genetically matched to the donor of the somatic cell (therapeutic cloning).

Epiblast Stem Cells (EpiSCs)

As described in Chapter 6, one of the first stages of postimplantation development is the separation of the ICM into two lineages, the hypoblast and the pluripotent epiblast.

Pluripotency can be considered as two states: newly segregated epiblast cells contain ‘naïve’ or ground state pluripotency, and following implantation these respond to signals from the extraembryonic tissues to become primed for differentiation. The two types of cells are characterized by expression of core pluripotency factors Oct4, Sox3 and Nanog, but they differ in their expression of other genes and their culture requirements.

Experiments with mouse embryos established that the epiblast in the late blastocyst is functionally and molecularly distinct from blastomeres, and from the ICM (Nichols and Smith, 2009). A strategically important milestone was reached in 2007, with the isolation of primed pluripotent stem cell lines derived from postimplantation (E5.5 to E6) mouse and rat epiblast (Brons et al., 2007; Tesar et al., 2007). Intriguingly, these EpiSCs differ significantly from mouse ESCs, but have key features in common with human cells. The two factors required for mouse ESC derivation (LIF and/or BMP4) have no effect on epiblast cell isolation, a similar situation to human ESC derivation. Instead, the signaling factors that are important for human ESCs (FGF and Activin/Nodal) are apparently critical for EpiSC derivation. The pattern of gene expression by EpiSCs differs from that of mouse ESCs, but they do retain the ability to proliferate indefinitely, as well as the potential for multilineage differentiation. Mouse ES cells can be induced to become EpiSCs, and the reverse transition has also been observed (Bao et al., 2009). It is possible that the unique properties that hESCs have in common with EpiSCs could reflect a different origin that had not previously been recognized in hESCs; i.e., they may represent a later stage of development (postimplantation epiblast) than mouse ES cells. Activin/Nodal signaling seems to have an evolutionarily conserved role in the maintenance of pluripotency. This also reinforces the significant species-specific differences in genetic and signaling pathways for lineage specification between humans and rodents. Several key TGF-ß signaling pathways were found to be enriched and differentially expressed in the human EPI and TE, and inhibiting this pathway downregulated Nanog expression in human, but not in mouse EPI cells – TGF-ß signaling is required for the development of pluripotent EPI in human blastocysts (Roode et al., 2012). The differences between species in expression of early lineage-specific genes have significant implications for developmental control and stem cell derivation.

Induced Pluripotent Cell Lines (IPSCs/PiPSCs)

The concept of reversing the programming of differentiated tissues to pluripotent states was introduced with the first somatic cell nuclear transfer (SCNT) experiments by John Gurdon during the late 1950s, using Xenopus oocytes (Gurdon, 1962). Several decades later, the birth of ‘Dolly the Sheep’ provided the first confirmation in mammals that a differentiated somatic cell could be converted to a totipotent state by inserting its nucleus into an enucleated oocyte. Dolly was born at the Roslin Institute in Edinburgh on July 5, 1996. The somatic cell nucleus was taken from a mammary gland cell and transferred into an unfertilized ovine oocyte that was then stimulated to divide by electrical pulse activation; the hybrid cell developed to blastocyst stage and was transferred to a recipient surrogate mother. Dolly died on February 14, 2003 from a progressive lung disease, 5 months before her 7th birthday.

With accumulated information regarding pluripotency in ESC, pinpointing key genes that might be used to reprogram somatic cells became a major research goal. Panels of genes that are enriched in ESC populations and thought to be involved in maintenance of pluripotency were screened, with the target of identifying factors that have the ability to reprogram somatic mouse cells into proliferation. From a panel of 24 target genes, four factors were found that together induced a transformation of mouse fibroblasts into cells that closely resemble mouse ES cells: OCT4, SOX2, c-Myc and Klf4 (Takahashi and Yamanaka, 2006). The experiments were conducted by using retroviruses to insert key pluripotency genes into the fibroblasts; the cells that resulted had properties analogous to ESCs in culture and also formed teratomas when injected into mice. The same technique was subsequently applied to successfully reprogram human fibroblasts into hES-like cells (Takahashi et al., 2007a; Lowry et al., 2008). A further independent study using human cells screened a panel of 14 genes that are enriched in hESCs and succeeded in reprogramming human fibroblasts with the introduction of genes for OCT4, SOX2, NANOG and LIN28 (Yu et al., 2007). Human iPS cells satisfy all the original criteria proposed for characterization of hESCs: morphology is similar, they express typical hESC surface antigens and genes, differentiate into multiple lineages in vitro, and when injected into SCID mice they form teratomas containing cells derived from all three primary germ layers. Since this initial report, numerous combinations of somatic cell type and cocktails of factors have been studied and continue to be investigated: some cell types can be reprogrammed more efficiently than others, and different types of cell respond to different expression levels and combinations of factors. The number of factors investigated continues to grow, and the original four now only head a list of related proteins that seem to enhance the efficiency of transformation in some cells (Heng et al., 2010). Efforts to elucidate the mechanisms by which such a limited number of transcription factors can erase and reprogram a differentiated state continue: the DNA-binding sites of OCT4, SOX2 and NANOG have been studied, and it seems that these three factors can also activate or repress the expression of many other genes, including transcription factors that are important during early stages of development (Boyer et al., 2005).

Spermatogonial Stem Cells (SSC)

As described in Chapter 3, primordial germ cells differentiate into type A (A0) spermatogonia in the fetal testis, and spermatogonia in the seminiferous tubules differentiate into mature sperm after puberty. Type A spermatogonia in the basal compartment of the tubules divide by mitosis to form two cells, another parent type A cell and a second (type B) diploid cell that is irreversibly committed to entering meiosis in order to start the journey toward mature haploid sperm cell production. Spermatogenesis continues throughout the life of a normal adult male, producing millions of sperm each day. The relatively small population of self-renewing type A spermatogonia are known as spermatogonial stem cells (SSCs), and these adult tissue stem cells are essential for male fertility, balancing self-renewal with differentiating divisions for continuous sperm production. hSSCs are unipotent stem cells; despite stages of amplification and differentiation, they ultimately produce only one cell type: mature sperm.

Their active proliferation makes them highly susceptible to damage by chemotherapy and radiation, and therefore patients undergoing treatment for cancer may become temporarily or permanently infertile. Semen and/or testicular biopsy cryopreservation have long been offered to men prior to initiation of gonadotoxic treatment; this option is not possible for prepubertal boys, who have not yet started spermatogenesis. The potential of using SSCs as a means of preserving a patient’s fertility remains an active focus for research directed toward understanding:

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   1. How SSCs keep their germline identity, and their paternal-specific pattern of epigenetic imprinting

   
   
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   2. The testicular niche and the intercellular communications that balance self-renewal with differentiation in order to allow lifelong gametogenesis.

In 1994, Brinster and colleagues described transplantation of SSCs from fertile donor mice to the testes of infertile recipients; the recipient mice initiated donor-derived spermatogenesis and produced functional sperm (Brinster et al., 1994). Since that time, experimental SSC transplantation has been used to restore fertility, with resulting embryos or offspring, in mice, rats, goats, sheep and monkeys (Gassei & Orwig, 2016). Based upon these experiments, many centers are now cryopreserving testicular tissue or cells from prepubertal boys before they start cancer treatment, in the hope that SSC-based therapies will be available in the future. Experimental strategies under investigation to achieve in-vivo or in-vitro spermatogenesis include testicular tissue grafting, testicular tissue organ culture, SSC culture and transplantation, and iPSCs.

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