Embryonic Stem Cells – Stem Cells and the Future of Regenerative …

Embryonic stem cells (ESCs) are found in the inner cell mass of the human blastocyst, an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. In normal embryonic development, they disappear after the 7th day, and begin to form the three embryonic tissue layers. ESCs extracted from the inner cell mass during the blastocyst stage, however, can be cultured in the laboratory and under the right conditions will proliferate indefinitely. ESCs growing in this undifferentiated state retain the potential to differentiate into cells of all three embryonic tissue layers. Research involving human ESCs is at the center of the ethical debate about stem cell use and potential in regenerative medicine. Embryos from which ESCs are extracted are destroyed in the process.

Several scientific questions are important when considering the potential of stem cells for use in regenerative medicine and the policy and ethical issues that arise:

What properties of ESCs have promise for regenerative medicine?

What direct evidence supports ESCs' effective use in regenerative medicine?

What obstacles and risks are associated with the use of ESCs in regenerative medicine?

Human ESCs were successfully grown in the laboratory for the first time in 1998 (Thompson et al., 1998). Under appropriate culture conditions, ESCs have demonstrated a remarkable ability to self-renew continuously, that is, to produce more cells like themselves that are multipotent. As indicated at the workshop by Thomas Okarma and Ron McKay, ESC lines established from single cells have been demonstrated to proliferate through 300-400 population-doubling cycles. Human ESCs that have been propagated for more than 2 years also demonstrate a stable and normal complement of chromosomes, in contrast to the unstable and abnormal complement of embryonic cancer cell lines used in the past to study early stages of embryonic development. Careful monitoring of the aging ESC lines will be needed to evaluate the significance of genetic changes that are expected to occur over time.

Because human ESCs have only recently become available for research, most of what is known about ESCs comes from studies in the mouse, which, as noted in Chapter 2, cannot be presumed to provide definitive evidence of the capabilities of human cells.

Nevertheless, ESCs derived from mouse blastocysts have been studied for 2 decades and provide a critical baseline of knowledge about the biology and cultivation of these cells (Torres, 1998; Wobus and Boheler, 1999). The factors that permit the mouse ESC to continue replicating in the laboratory without differentiation and methods to trigger differentiation into different cell types that exhibit normal function have been actively explored. Among the types of cells derived from cultured mouse ESCs are fat cells, various brain and nervous system cells, insulin-producing cells of the pancreas, bone cells, hematopoietic cells, yolk sac, endothelial cells, primitive endodermal cells, and smooth and striated muscle cells, including cardiomyocytesheart muscle cells (Odorico et al., 2001).

Experience with mouse ESCs has provided clues to methods for culturing human ESCs and leading them to differentiate. Mouse ESCs will proliferate in an undifferentiated state in the presence of a biochemical called leukemia inhibitory factor (LIF), but the culture conditions required to keep human ESCs from differentiating include growing them in petri dishes on a layer of mouse embryonic fibroblasts (referred to as feeder cells) in a medium containing serum from cows. The feeder cells are inactivated, so they are not dividing and expanding, but they produce growth factors that sustain the ESCs. The mechanism of how feeder cells maintain the proliferation of undifferentiated ESCs is unknown. Such in vitro culturing presents certain theoretical hazards to the use of stem cells for regenerative medicine, such as the spread of viruses and other infectious agents not normally found in humans. When removed from feeder cells and grown in suspension (in liquid), human ESCs form aggregated balls of cells called embryonic bodies, which have been reported to give rise to a multiplicity of cell types representing all three layers of embryonic tissue development (Itskovitz-Eldor et al., 2000; Reubinoff et al., 2000; Schuldiner et al., 2000). Evidence of the differentiation in culture includes detection of the products of genes associated with different cell types and in some cases by the characteristic shapes that are peculiar to different cell types. Cells derived from human embryonic bodies include rhythmically contracting cardiomyocytes, pigmented and nonpigmented epithelial cells, and neural cells displaying an exuberant outgrowth of axons and dendrites (Odorico et al., 2001). In other experiments, cells arising from human ESCs have been reported to express genes associated with liver and pancreas function (Schuldiner et al., 2000). Human ESCs grown in coculture with mouse bone marrow stromal cells have been reported to produce colonies of human hematopoietic precursors and ultimately cells from the blood (Kaufman et al., 1999).

Further evidence of the multipotent capability of human ESCs is based on studies in an in vivo setting. Human ESCs injected into mice form a type of benign tumor called a teratoma that is made up of tissues from all three embryonic layers. The tissues that arise in the tumor are often advanced, organized, and complex, and include teeth, gut, hair follicles, skin, epithelium, muscle, bone, cartilage, lung tissue, and neural cells (Thompson et al., 1998). The experiments showed the capability of ESCs to produce a variety of tissues, but the results also highlight the complexity of the biological program of tissue development that can unfold in different biological environments. These results also emphasize the abnormal, potentially neoplastic potential of ESCs when placed into unnatural environments.

Major questions remain about the genetic or environmental factors in the body that control the fate of ESCs and about the importance of different factors during various stages of cell differentiation. Even on the basis of the limited findings, however, the ability to grow human ESCs in vitro and to have them differentiate in the laboratory makes them an important and unique tool with which to conduct the basic research that is critical for the foundation of future regenerative therapies. It has been possible, for example, to create a lineage of mouse ESCs that generate neural cell precursors (Li et al., 1998). Studies of the genes turned on and off as cells begin to differentiate, which are already under way with ESCs, will permit a better understanding of the genetic controls important in tissue differentiation (Duncan et al., 1998). In vitro studies of ESCs also provide an opportunity to explore the role of biochemicals produced in the normal cellular environment that induce stem cells to differentiate, to migrate to a site needing repair, and to assimilate into tissues (Schuldiner et al., 2000).

At the workshop, James Thomson and Thomas Okarma suggested that human ESCs will someday provide a potentially unlimited source of cells, differentiated in vitro, for transplantation therapies involving the liver, nervous system, and pancreas. Irving Weissman alluded to the possible use of ESCs to enhance the success of whole-organ transplantation. If HSCs derived from human ESCs could be successfully transplanted into the blood system of a transplant recipient (by using immunosuppressive drugs), any further implant tissue (say kidney or pancreas) developed with the same ESCs would not, in theory, be rejected by the recipient because the immune cells produced in the recipient's blood by the HSCs would see the implant tissue as self.

But that is a long way off, as Marcus Grompe noted, in as much as no one has yet demonstrated any in vivo reconstitution of an organ's function in either humans or experimental animals with cells derived from human ESCs. Moreover, ESCs in tissue culture give rise to a mixture of cell types all at once, and biochemical, tissue-culture, and molecular-biology techniques to control and limit differentiation require much further investigation.

Because human ESCs have only recently become available for research, and because public funding for such research has been limited, studies of how well ESCs or their differentiated tissues perform physiologic functions has been largely conducted with mouse models. Ron McKay described progress made in coaxing the in vitro differentiation of human ESCs into insulin-producing cells that might be useful in treating diabetes, but he also noted that studies have already been conducted with analogous mouse cells transplanted into mice that have diabetes and that partial restoration of insulin regulation was observed (Lumelsky et al., 2001). Other studies have demonstrated that mouse ESCs can be successfully transplanted into rodents that have Parkinson's disease symptoms and partially relieve these symptoms (Studer et al., 1998). Similarly, studies suggest that mouse ESCs can be transplanted into animals that have spinal-cord injuries and partially restore neural function (McDonald et al., 1999).

Those studies provide promise, but not definitive evidence, that similar treatments could be effective in humans. Human ESCs will need to be tested in primate models, such as those for Parkinson's disease and diabetes mellitus in the rhesus monkey. Methods for transplanting ESCs need to be developed, as do means of establishing whether the cells develop and function properly after transplantation. In some cases, it will be important to ensure that the transplanted cells or tissues are incorporated and positioned properly relative to existing tissues, such as in heart and neural tissue; the three-dimensional, cell-to-cell interactions will play important roles in the functioning of an organ. Other cells, like pancreatic islet cells, or hematopoietic cells, will require less complex incorporation.

Also, the large-scale propagation of human ESCs in culture will require that they can be grown without feeder cells (Odorico et al., 2001). Research is needed to elucidate the mechanisms of feeder cells in repressing differentiation and to find alternatives to them, at the same time eliminating the potential that an animal virus from the feeder cells might be transferred to the ESCs.

Finally, it was noted earlier that the chromosomes of human ESCs have been shown to be stable in tissue culture. This does not mean however, that ESC lines will not be subject to the random mutations that affect all cell lines as they age. In cells from humans and other animals, approximately one mutation occurs every time a cell divides. A cell that has divided 200 times in culture therefore can be expected to harbor approximately 200 different mutations (Kunkel and Bebeneck, 2000). So far, there have been no studies published about the changes that may have occurred in existing stem cell lines. Vigilant monitoring of the integrity of existing cell lines is essential to allow understanding of the impact of long-term culture, and new stem cell lines may need to be developed in the future.

In addition to demonstrating the functional effectiveness of ESC transplants, it is necessary to identify and minimize, or eliminate, the risks that ESCs might pose. Two identifiable risks are tumor formation and immune rejection. As noted earlier, human ESCs injected into mice can produce a benign tumor made up of diverse tissues; this response is believed to be related to the multipotency of the undifferentiated cells in an in vivo environment. However, in a small number of short-term studies in mice, human ESCs that have been allowed to begin the process of differentiation before transplantation have not resulted in significant tumor formation (Odorico et al., 2001). Obviously, this is a critical problem to understand and control.

It is too early to tell, therefore, whether it will be appropriate to use human ESCs directly in regenerative medicine. A great deal obviously must be elucidated about how the body controls the differentiation of stem cells, and this has yet to be reliably reproduced in vitro. Also, the behavior of ESCs implanted in a specific organ has not been well studied. It might someday be possible to add growth factors with a transplant to stimulate the production of a particular cell type or multiple cell types. Inducer tissues that interact with stem cells might be cotransplanted with ESCs to achieve a similar result. Those possibilities are still in experimental investigation.

In another respect, the possible problems associated with ESC transplantation are common to all transplantation, such as the risk of infection and the risk of tissue rejection. As discussed in Chapter 2, rejection is a serious obstacle to successful transplantation of stem cells and tissues derived from them. It has been suggested that ESCs provoke less of an immune reaction than a whole-organ transplant, but it is unclear whether that will be true of the regenerated tissues derived from ESCs. Some types of cells (such as dendritic cells, immune system cells, and vascular endothelial cells) carry more of the histocompatibility antigens that provoke immune reactions than other cells. Those types are present in the tissues of whole organs; they connect an organ with the bloodstream and nervous system. However, tissue derived in vitro from ESCs, such as liver tissue, would not contain such cells and therefore would theoretically trigger a milder immune response; this assumes that techniques for controlling differentiation of ESCs will be available. In addition, the liver cells likely would not be devoid of all surface antigens, and so, in the absence of other techniques to reduce transplant rejection, the use of immunosuppressive drugs will still have to be used, with attendant risks of infection and toxicity.

Although difficult to conceive, the creation of a very large number of ESC lines might be one way to obtain a diversity of cells that could theoretically increase the chances of matching the histocompatibility antigens of a transplant recipient. It has also been suggested that ESCs could be made less reactive by using genetic engineering to eliminate or introduce the presence of surface antigens on them (Odorico, 2001). An exact genetic match between a transplant recipient and tissue generated from ESCs could also, in theory, be achieved by using somatic cell nuclear transfer to create histocompatible ESCs (). Cells created with this technique would overcome the problem of immune rejection. However, it might to not be appropriate to transplant such cells in a person with a genetically based disease, since the cells would carry the same genetic information. In any case, an understanding of how to prevent rejection of transplanted cells is fundamental to their becoming useful for regenerative medicine and represents one of the greatest challenges for research in this field.

Somatic Cell Nuclear Transfer (SCNT)

Visit link:
Embryonic Stem Cells - Stem Cells and the Future of Regenerative ...

Human embryonic stem cells: Derivation, culture, and differentiation: A …

The ectoderm derivatives include external ectoderm, neural crest, and neural tube. These structures give rise to cells of the epidermis, external sense organs, and the peripheral and central nervous system (Gilbert, 2006).

Generating functional neurons from hESC with the aim of treating neurodegenerative diseases is the subject of intensive investigation. Shortly after derivation of the first hESC lines, Reubinoff et al. (2000) described isolation of expandable neural progenitor cells from hESC that had been cultivated for four to seven weeks at a high density in vitro. The authors illustrated that the neuroepithelium contained areas of differentiating hESC colonies, identified by expression of the embryonic polysialylated neural cell adhesion molecule (PSA-NCAM), and had distinct morphological features. These areas were mechanically dissected and expanded as neural aggregates or spheres in serum-free media. Neural induction was achieved by plating the spheres on coverslips coated with poly-D-lysine and laminin, which resulted in emergence of cells expressing the neuronal markers -tubulin and microtubule-associated protein 2 (MAP2). They also identified a subset of the neuronal cells as being glutaminergic and GABAergic neurons, as shown by the expression of glutamate and glutamic acid decarboxylase (GAD).

Reubinoff and coworkers (2001) also optimized the expansion of hESC-derived NPC by addition of B27 supplement, human recombinant epidermal factor EGF and the mitogen bFGF. The expanded NPC were capable of differentiating into all three major neural lineages (neurons, astrocytes, oligodendrocytes) in vitro as well as in vivo. Lineage tracing studies showed that NPC grafted to the ventricles of newborn mice differentiated in a region-specific manner, according to normal developmental patterning signals. For example, neuronal differentiation was specifically detected in the olfactory bulb, where postnatal neurogenesis occurs (Reubinoff et al., 2001).

At about the same time, Zhang et al. (2001) used a different protocol with similar success in generating enriched populations of NPC from hESC. In that study, differentiating EBs were treated with insulin, transferrin, progesterone, heparin, and bFGF. Continuous exposure to bFGF led to formation of monolayers of neural tube-like rosettes that were isolated by dispase enzymatic treatment. Similar to the study by Reubinoff et al. (2001), the generated NPC were capable of generating oligodendrocytes, astrocytes, and mature neurons both in vitro and after transplantation into mice.

These observations confirming the multi-lineage differentiation potential of hESC-derived NPC, and promising indications of survival and integration of these cells in vivo, set the stage for future developments of methods for selective differentiation of different neuronal phenotypes that could potentially be used to treat several central nervous system disorders. Indeed, studies examining the signals and factors that govern the proliferation and cell fate specification of neural progenitors are accumulating rapidly.

Generation of transplantable motor neurons from hESC could have potential for treating victims of spinal cord injuries, or degenerative diseases such as amyotrophic lateral sclerosis. The first functional motor neurons originating from hESC were described by Li et al. (2005). In that study, Li and colleagues used the previously described method (Zhang et al., 2001) to generate NPC cells, which were subsequently induced to become motor neurons by addition of RA to the culture medium. Further maturation to postmitotic motorneurons was induced by the ventralizing morphogenic protein SHH. An interesting aspect of this study was the specific temporal effect of RA on motor neuron induction, in that RA could induce only early, but not late neuroectodermal cells, to differentiate into motorneurons (Li et al., 2005). This occurred through upregulation of expression of HOX genes that are involved in assigning the rostrocaudal positional identity of spinal motor neurons. The functionality of the generated motor neurons was confirmed by electrophysiological experiments and establishment of neuromuscular transmission in motorneuron-myotube co-cultures (Li et al., 2005).

In another study, directed differentiation of motor neurons was achieved by addition of RA/SHH extrinsic cues to cultures of differentiating NPC generated from hESC (Lee et al., 2007). With respect to clinical potential, transplantation of hESC-derived motor neurons in developing chick embryo spinal cord showed that these cells were capable of survival and directed axonal growth over relatively long distances (Lee et al., 2007). Nevertheless, transplantation in adult rats did not result in axonal growth to outside the CNS. It should be mentioned that, although caudal and ventral patterning was achieved by RA and SHH, the initial neural induction of hESC was obtained by co-culture with MS5 mouse stromal cells, which would preclude the use of motorneurons generated by this particular strategy for any type of human transplantation therapy (Lee et al., 2007).

In a later study by Li et al. (2008), neural induction medium containing heparin and cyclic adenosine monophosphate (cAMP) in addition to RA and SHH was successful in generating a nearly homogenous population of ventral spinal progenitor cells, with highly efficient generation of motor neurons.

Promoting remyelination for treatment of neurologic disorders caused by demyelination of motor neurons is another potential application of hESC-derived cells. One of the strategies used to promote remyelination involves transplantation of oligodendrocytes, which produce the myelin sheath of motor neurons and are essential for normal signal conduction. In 2005, Keirstead and his associates demonstrated that transplantation of hESC-derived oligodendrocyte progenitor cells (OPC) produced by glial restriction media, can lead to remyelination of motorneurons, and recovery of motor function after spinal cord injury in rats (Keirstead et al., 2005). Following further assessment of the safety concerns associated with OPC transplantation in animal models (Cloutier et al., 2006), Geron Corporation obtained FDA clearance in January 2009 to begin the first human clinical trials of hESC-derived cells in the United States (see Alper 2009). OPC were generated from the H1 hESC line under current good manufacturing practices without the use of feeder cells, in defined media containing only human recombinant proteins. The Phase I trial is designed to assess the safety of transplantation of OPC in patients with acute thoracic spinal cord injuries and will be carried out at multiple medical centers.

A number of additional studies have reported the production of multiple neuronal subtypes, including cholinergic, serotonergic, GABAergic, and dopaminergic (DA) neurons, from hESC (Erceg et al., 2008; Gerrard et al., 2005; Perrier et al., 2004; Yan et al., 2005). As previously mentioned, protocols used for neural conversion of hESC generally give rise to a mixture of neuronal phenotypes. Previous studies of neural differentiation of mouse ESC have established protocols for growth factor-mediated lineage selection and survival-promoting factors of neuronal cells (Barberi et al., 2003; Lee et al., 2000; Okabe et al., 1996). As a general strategy for obtaining selective neuronal differentiation, factors with effects on the anteroposterior (AP) or the dorsoventral (DV) neuronal patterning in combination with specific neurotrophins are used at specific stages during in vitro ESC differentiation. In a comprehensive study of neural development of mouse ESC, Barberi et al. used a stromal feeder-based differentiation system to generate early ectodermal cells (6 days co-culture) and identified various combinations of factors that govern neural and neuronal subtype specification () (Barberi et al., 2003). It should be noted that these differentiation strategies for mouse ESC cannot be directly applied to hESC without some modifications.

Neural subtype specification from neural progenitors derived from mouse ESC using various combinations of inducing factors. AA, ascorbic acid; bFGF, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; FGF4, fibroblast growth factor 4; FGF8, fibroblast growth factor 8; NT4, neurotrophin-4; PDGF, platelet-derived growth factor; RA, retinoic acid; SHH, sonic hedgehog. {Adapted from Barberi et al. (2003), [100]}.

To date, the majority of studies on neural differentiation of hESC have been focused on generation of dopamine producing neurons of the midbrain subtype, due to their potential application in cell replacement therapy for Parkinsons disease. Established protocols used to generate DA neurons include allowing spontaneous differentiation of hESC, followed by addition of DA inducing molecules, SHH and FGF8, and later neurotrophic factors, or by culturing hESC on feeder cells from animal or human origin that have the ability to direct hESC to become DA neurons.

Kawasaki and coworkers in Japan discovered in 2000 that certain mouse stromal cell lines had a neural and DA promoting effect on mouse ESC (Kawasaki et al., 2000). The authors showed that the activity of the stromal cells was not mimicked by FGF8/SHH, or Wnt signaling, previously known to be key factors in development and patterning of midbrain DA neurons. Thus, this strategy was established as a new approach to generate DA neurons and was termed stromal-derived inducing activity (SDIA).

Our group as well as others have adapted this approach to generate DA neurons from hESC. When the hESC line BG01 was cultured on the mouse stromal cells for three weeks, approximately 87% of colonies contained large numbers of TH+ cells (Zeng et al., 2004). The TH+ neurons generated by SDIA had midbrain characteristics, as determined by expression of Nurr1 and Pitx3 transcription factors that are strongly associated with midbrain DA neurons. The DA neurons were functional in vitro as confirmed by electrophysiological assessments and release of dopamine. However, the survival of TH+ neurons grafted into the striatum of parkinsonian rats was very limited. A parallel study of DA induction of hESC by Perrier and colleagues (2004), combined SDIA with SHH and FGF8 patterning molecules, ascorbic acid, and various neurotrophic factors including BDNF, GDNF, TGF-3, dcAMP and demonstrated that the yield and functional properties of TH+ neurons were highly dependent on exposure to SHH and FGF8.

Other feeder cells that possess DA-inducing activity, and that have been used to generate DA neurons from ESC, include testis-derived sertoli cells (Yue et al., 2006), meningeal cells (Hayashi 2008), and striatal or mesencepahlic astrocytes (Buytaert-Hoefen et al., 2004; Roy et al., 2006). Secreted factors produced by astrocytes have also been reported to promote neurogenesis and induction of DA neurons (Nakayama et al., 2003).

Yan and coworkers (2005) demonstrated neural and DA induction of hESC in the absence of any type of feeder cells by addition of SHH and FGF8 to EB-derived neural rosettes which were manually isolated from mixed cultures. The resulting TH+ neurons comprised 50%60% of the total neuronal population and were electrophysiologically active. Other differentiation paradigms have included addition of an NPC expansion step to this protocol in order to generate a more pure population of DA neurons (Cho et al., 2008).

Although mouse stromal cells that possess SDIA activity are considered as one of the most efficient tools for converting hESC to DA neurons, the use of animal cells would preclude any downstream clinical application due to possible transfer of xenogeneic material. To understand the molecular activity of SDIA, we further assessed the activity of stromal cells and found that stromal cell surface activity promoted hESC survival and was able to enhance overall neurogenesis, whereas soluble secreted factors provided DA lineage-specific instructions (Vazin et al., 2008). We then examined the gene expression profile of potent PA6 stromal cells as compared to that of cell lines lacking the DA-inducing effect (Vazin et al., 2009). Several soluble factors and growth-inducing proteins potentially responsible for the DA phenotype-promoting component of SDIA were identified, based on high levels of expression in potent DA-inducing PA6 cells. Testing of these factors showed that a combination of four factors, stromal cell-derived factor 1, pleiotrophin, insulin-like growth factor 2, and ephrin-B1, termed SPIE was sufficient to induce DA neuronal differentiation from hESC. The combination of these four factors mimicked SDIA activity, providing an approach for differentiating DA neurons from hESC in a culture system that is potentially suitable for clinical applications (Vazin et al., 2009).

Transplantation of DA precursors or neuronal cells is still at the stage where survival and integration needs to be optimized, as the majority of studies focusing on neural transplantation have reported limited or no survival of DA neurons. A few studies have, however, reported more encouraging results. A study by Roy et al. (2006) transplanted hESC-derived DA progenitors induced with immortalized human fetal midbrain astrocytes in the presence of SHH and FGF8, and illustrated that about 21% of the total number of transplanted cells (5 105 cells) were TH+. Long-lasting behavioral recovery was found in animals that received cell implants. The enhanced viability of the TH+ neurons post-transplantation may have been caused by the influence of fetal midbrain astrocytes during development or specification of these neurons.

A more recent study by Chiba and colleagues (2008) has indicated that SDIA-induced DA differentiation of hESC can be improved by addition of the BMP inhibitor noggin. Importantly, the number of TH+ cells found in animals transplanted with hESC treated with noggin was five times more (average of about 500 cells/animal) than the animals that received hESC induced by SDIA alone. The enhanced in vivo viability of TH+ cells was also reflected in animal behavioral recovery.

As previously discussed, the patterning of the neural tube along its DV and AP axis is determined by specific concentrations of morphogens including SHH, BMP, FGF and RA. Other important aspects involved in regional specification of NPC are the temporal effect of these factors, as well as the duration of signaling. There is evidence indicating that NPC progressively lose their differentiation potential and can no longer be regionally specified in response to instructive patterning cues after extended in vitro culturing (Machon et al., 2005; Santa-Olalla et al., 2003).

A recent study by Elkabetz and colleagues (2008) has identified a novel population of hESC-derived neural stem cells with a unique gene expression profile, termed neural rosette cells (R-NSC), which are isolated at an earlier stage of differentiation, as compared to the previously described NPC. Forse1 was used as a marker to isolate these early rosette stage cells, which adopted an anterior forebrain characteristic in the absence of extrinsic patterning factors. In contrast to NSC, the R-NSC could be re-specified toward caudal neuronal fates including motor neurons and midbrain DA neurons by SHH/RA and SHH/FGF8 treatment, respectively. This study also illustrated the in vivo survival and phenotype maintenance of these two rosette stage-derived neuronal phenotypes.

These findings provide evidence that neuronal plasticity of NSC is highly dependent on the developmental stage and restricted to a specific time window. Selective expansion of neural stem cells that retain their ability to differentiate towards specified neurons is of great potential value. Moreover, generation of restricted NSC has clinical relevance, as such cells have been reported to have a tendency to migrate towards the site of injury and rescue degenerating neurons following implantation in animal models (Bjugstad et al., 2008; Ourednik et al., 2002).

Characterization studies of SDIA-mediated neural induction have also suggested that midbrain regional identity can only be established during early stages of ESC differentiation (Parmar and Li, 2007). In addition, it has been suggested that early exposure of FGF8, before the onset of the neural stem cell transcription factor Sox1, is necessary for generation of DA neurons with a midbrain phenotype (Yan et al., 2005). Signaling duration is also known to affect the mechanisms that underlie the patterning role of factors. For example cells are known to respond analogously to varying concentrations of SHH, or to varying duration of exposure to this factor (Dessaud et al., 2007).

See the rest here:
Human embryonic stem cells: Derivation, culture, and differentiation: A ...

Controversy Over the Use of Embryonic Stem Cells in Research Hadron

Written by Margaret Wei

Stem cells have been increasing in popularity in research in recent years due to their pluripotency. Stem cells are at first unspecialized, but have the capacity to develop into specialized cells hence their valued versatility when it comes to research. Stem cell research is used to increase understanding of how diseases occur. Researchers do this by observing how the stem cells differentiate into the specialized cells (bones, heart muscle, nerves, and other organs and tissue) to better determine how diseases and conditions develop.

Origin of Stem Cells

Several different types of stem cells are used in research: embryonic, adult, induced pluripotent, and perinatal. Embryonic stem cells originate from donated fertilized eggs in vitro fertilization clinics but never implanted in a womans uterus. Adult stem cells are derived from tissue, like bone marrow, and can be used to grow different types of specialized cells. Induced pluripotent stem cells are manipulated through genetic reprogramming to transform adult cells to stem cells, and then they can be used to replace dysfunctional cells. Perinatal stem cells come from amniotic fluid and can develop into specialized cells. In laboratories, stem cells can live and grow in special solutions in test tubes or petri dishes

Of the different types of stem cells, embryonic stem cells are the most controversial because their research consists primarily of experimentation on cells that have the potential to become a developed human. These cells are derived from embryos at a developmental stage before implantation would normally occur in the uterus. During this time, fertilization occurs in the oviduct, and over the span of the next few days, the cell divides multiple times as it travels to the uterus. At this point, the embryonic cells are undifferentiated, in other words, they do not look or act like the specialized cells of the adult, and they have the potential to become any specialized adult cell type (Yu, J. and Thomson, J.A.).

The first stage of differentiation occurs after five days, and an outer layer of cells that was supposed to become a part of the placenta separates from the inner cell mass (ICM). Because of the process of implantation, the ICM cells no longer have the potential to develop into any cell type of the body, and they are quickly depleted as they differentiate into limited types of cells. Eventually, the ICM-derived cells are fixed to be embryonic stem cells if the ICM is removed from its normal embryonic environment and cultured under appropriate conditions. These specific conditions allow the cells to proliferate and replicate, while maintaining the developmental potential to form any cell type of the body (Yu, J. and Thomson, J.A.).

The diagram above illustrates the process of obtaining embryonic stem cells.

Interactive resources for schools. (n.d.). Retrieved December 30, 2020, from https://www.abpischools.org.uk/topic/stem-cells/4

Embryonic Stem Cells in Research

Stem cells are currently used in regenerative medicine where healthy cells are generated to replace diseased cells. Scientists use embryonic stem cells to determine how specific cells can be used to regenerate and repair diseased or damaged tissues in people. Further research on stem cell regeneration would be beneficial to those who have Parkinsons disease, Alzheimers disease, heart disease, stroke, burns, cancer, etc. The potential that stem cells have to become new tissue can be used in transplant and regenerative medicine.

To ensure that drugs are safe to be used on people, researchers use stem cells to test drug effectiveness before it gets approved for drug development for cardiac toxicity testing. The stem cells that are to be tested on must be programmed to acquire properties of the type of cells targeted by the drug, but more research needs to be done about how to program cells into specific cells to produce more accurate results from the tests that would show what kind of effect the drug had on the cells (Railton, 2019).

Stem Cell Controversy

Despite the benefit of the pluripotency of embryonic stem cells, controversy arises behind how embryonic stem cells originate. Because embryonic stem cells are extracted from human embryos, many scientists question the ethics of embryonic stem cell research because it is correlated to human testing. Additionally, many disagree with testing on embryonic stem cells because the fertilized embryo has the potential to develop into a human. Concerns arise about what qualities determine humans along with the pervasive debate of what constitutes as the official beginning of human life during embryonic development.

According to certain faiths and religions, human life begins at conception, meaning that an embryo is equal to a person and it has the same rights that must be respected. As a result, the scientific process of extracting the embryonic stem cell from a blastocyst and removing the inner cell mass is amounting to murder (Lo, B., & Parham, L., 2009). This mindset is commonly correlated to the opposition of abortion and with the pro-life movement. However, a number of pro-life leaders support stem cell research using frozen embryos that remain after a woman or couple has completed infertility treatment and that they have decided not to give to another couple.

According to Senator Orrin Hatch, a former U.S. senator and a representative of the conservative party interposed the idea that, I believe that human life begins in the womb, not a Petri dish or refrigerator To me, the morality of the situation dictates that these embryos, which are routinely discarded, be used to improve and save lives. The tragedy would be in not using these embryos to save lives when the alternative is that they would be discarded.

A moderate view that others possess in terms of the morality of the embryo would be that the embryo would develop into a person later in development, well after fertilization. This view believes that a newly fertilized embryo does not constitute the same characteristics as a developed individual. However this does not always mean that they do not fully believe that embryonic stem cells are solely a clump of cells and that it is ethical for research without restriction. This does not mean that they do not agree with embryonic stem cell research, many hold a middle ground in which the embryo deserves to be perceived as a potential human being, but it is acceptable to use for certain types of research given good scientific justification, careful oversight, and informed consent from the embryo donor for research (Lo, B., & Parham, L., 2009).

Solutions to Reduce Controversy

Due to the rising controversy on this topic, The National Institutes of Health created guidelines for human stem cell research in 2009, which stated how they may be used in research. Also, to ensure that ethics are not violated, the guidelines state embryonic stem cells from embryos created by in vitro fertilization can be used only when the embryo is no longer needed.

Additionally, to reduce the controversy surrounding the idea of the potential of embryonic stem cells to develop into a human, scientists began to use pluripotent stem cells as an alternative. Since pluripotent stem cells are genetically reprogrammed to transform adult cells into stem cells, they do not have the same ethical concerns as embryonic stem cells. Therefore, genetically reprogrammed pluripotent stem cells do not have the same ethical concerns as embryonic stem cells in terms of origin.

References

Lo, B., & Parham, L. (2009, May). Ethical issues in stem cell research. Retrieved December 27,

2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726839/

Orive, G., Hernndez R.M., Gascn A.R., Igartua, M., Luis Pedraz, J. (2003, Mar).

Controversies Over Stem Cell Research. Retrieved December 29, 2020, from https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(03)00003-9

Railton, D. (2019, Feb). Stem cells: Therapy, controversy, and research. (n.d.). Retrieved

December 27, 2020, from https://www.medicalnewstoday.com/articles/200904

Yu, J. and Thomson, J.A. Embryonic Stem Cells. (n.d.). Retrieved December 30, 2020, from

https://stemcells.nih.gov/info/Regenerative_Medicine/2006chapter1.htm

See the original post:
Controversy Over the Use of Embryonic Stem Cells in Research Hadron

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements – Nature.com

The DANIO-CODE DCC

We established a DCC protocol26, which we populated with zebrafish developmental genomic data, including standardized annotation of metadata of diverse, often inconsistently annotated, published datasets (Fig. 1a), by the DANIO-CODE consortium (https://www.birmingham.ac.uk/generic/danio-code/partners/index.aspx). The DCC is accessible from ZFIN and includes datasets, their underlying samples and sequencing protocols using ZFIN and ENCODE nomenclature (www.danio-code.zfin.org). To identify and analyze the developmental dynamics of genomic features, direct comparison across datasets produced by different laboratories and different protocols is required. To this end, we carried out consistent reprocessing starting from the raw sequencing data (Fig. 1a). Raw sequencing data were collected and reprocessed by standardized pipelines of ENCODE for ChIPseq and ATAC-seq27, FANTOM for CAGE-seq28 and producer pipelines for Hi-C, 4C-seq or other data (Methods). These pipelines are available on GitLab (https://gitlab.com/danio-code). The DCC data include 1,438 published datasets contributed by data producers directly or collected by DANIO-CODE data annotators, together with strategically selected datasets for developmental stages from the public domain. In addition, 366 datasets were generated by consortium members to fill gaps and to aid functional annotation and functional element characterization, including 15 CAGE-seq, 18 ChIPseq, 11 ATAC-seq, 2 Hi-C and 320 4C-seq datasets (Fig. 1b and Extended Data Fig. 1a,b). Breakdown of the datasets according to data types and stages of development is presented in Fig. 1b. The source of data collection is in Extended Data Figure 1c and Supplementary Table 1. Quality checks and data comparability analyses were carried out for datasets within a data type obtained from multiple laboratories, particularly affecting RNA-seq (Supplementary Fig. 1b), ChIPseq (Supplementary Fig. 1df), CAGE-seq (Supplementary Fig. 2) and ATAC-seq (Supplementary Fig. 1c) data. The DCC continues to be periodically updated (Extended Data Fig. 1e) and is openly accessible to the community for downloading data and uploading new datasets (Supplementary Videos 1 and 2).

a, Collection and manual annotation processes of datasets with the DANIO-CODE DCC with highlights of key findings. b, Extent of the open repository for developmental multiomic data for zebrafish with assay type (y axis) and developmental stage (x axis). Data first reported in this study are highlighted with black circles. c, Visualization of temporal dynamics of selected transcriptomic and epigenomic features during development at a developmentally active locus. Coloring of tracks represents developmental series from maternal (blue) to zygotically active stages of embryogenesis (red). Symbols and track colors indicate representative stages (Extended Data Fig. 1d). CNS, central nervous system.

The resulting data and reprocessed multiomic datasets represent a comprehensive annotation of the zebrafish genome during normal embryonic development and are available as a public track hub in the UCSC browser and uploadable to the Ensembl genome browser. Figure 1c provides an example developmentally regulated locus covering selected stages visualized by the Washington University Epigenome browser29. The tracks further include annotation of approximately 140,000 predicted ATAC-seq-supported developmental regulatory elements (PADRE) annotated by ChromHMM categories. The bulk sample-based predictions for regulatory elements are complemented with annotations of cell-type specificity of candidate regulatory elements provided by single-cell ATAC-seq30 (Supplementary Videos 35).

As genome-wide transcriptome analyses3,31,32,33 fail to annotate 5 untranslated regions (UTRs) precisely, we used DANIO-CODE expression data to improve current Ensembl models of developmentally active genes. We utilized 139 developmental RNA-seq samples to identify 31,458 genes comprising 55,596 transcripts (Fig. 2a and Supplementary Table 2), among them 167 novel transcripts of uncertain coding potential (TUCP) and 726 long noncoding (lnc) RNA genes not previously annotated by Ensembl and supported by CAGE signals (Extended Data Fig. 2 and Supplementary Table 3). We mapped 5 transcription start sites (TSSs) from 34 CAGE samples in 16 developmental stages (Fig. 2a). We applied promoter-calling criteria to CAGE data (Methods and Supplementary Fig. 2ac), resulting in 22,500 active promoters per CAGE sample on average, corresponding to 16,303 genes (Supplementary Table 4), and adding 4,070 novel promoters to 18,461 previously annotated Ensembl TSSs (GRCz10). To supplement the promoterome with cis-regulatory sites, we curated 581 regulatory motifs representing 814 zebrafish transcription factor (TFs), and predicted binding sites for these motifs across all promoters (Methods).

a, DANIO-CODE transcript 5 ends supported by CAGE TSS during stages of development. b, Distribution of absolute distance of Ensembl TSSs to CAGE-dominant TSSs in the Prim-5 stage. c, Relationship between guide distance to TSSs and ddCt. Inset: number of dCas guides for all 26 tested genes. d, CAGE-defined TSSs increase the accuracy of promoter identification and support dCas inhibition guide reagent designs. Distance between Ensembl TSSs and CAGE-dominant TSSs (top). Genome view with CRISPR guide position and efficacy, Ensembl and RefSeq transcripts, CAGE and RNA-seq expression (bottom).

a, Heat map shows the dynamics of expression levels of reference and alternative promoters across 16 developmental stages represented as images. Expression levels are scaled in the range of 01 for each row. Reference and alternative transcripts using the same and different coding sequence (CDS) starts are denoted. Transcript pairs without full CDS annotation are denoted as ambiguous. b, Distribution of correlation coefficient of expression levels of promoters across 16 developmental stages. c, Enrichment of KEGG pathways on multipromoter genes. The adjusted P value cut-off is 0.05, denoted by a vertical dashed line. The number of genes in KEGG pathways and those overlapping with multipromoter genes is shown inside the bars, d, MARA motif activity plots of three TF motifs across development. Posterior means and standard deviations (depicted as error bars) are based on analysis of the expression levels of all n=27,781 promoters for each sample. Motif logos are depicted as insets. e, Genome browser view of the actin alpha 1a promoter. From the top: ATAC signal, CAGE signal, a single TSR (black), two Ensembl transcripts (dark red) and TFBSs predicted to regulate this TSR (red) are shown. Color intensities of the TFBSs reflect MARA scores of predicted regulatory role of TFs.

Our above definition of promoters at single-nucleotide resolution may offer important guidance for promoter-targeted gene manipulation. For instance, gene promoter targeting for transcription block may be useful in reverse genetic experiments to avoid mutant RNA-mediated genetic compensation, which may mask mutant phenotypes and hinder dissection of gene function34. We compared Ensembls RNA-seq-based TSS with our CAGE-seq-based TSS and found a substantial discrepancy in position (Fig. 2b and Extended Data Fig. 3a), potentially impacting guide RNA design for CRISPRCas targeting. Multiple dCas guide positions were designed and their impact on expression reduction with increased distance between the guide target and dominant CAGE-defined TSS was tested. Efficiency of dCas inhibition was higher when CAGE dominant, compared to Ensembl, start sites were used (Fig. 2c,d and Supplementary Table 5), demonstrating the importance of accurate TSS detection and the improved accuracy of CAGE over the current Ensembl pipeline in promoter detection.

Using these data we identified 1,293 multipromoter genes (Supplementary Table 6), where 1,176 genes had one reference and one alternative promoter and 117 genes had two or more alternative promoters. Correlation of expression levels of reference and alternative promoter pairs indicated both convergent (cyan in Fig. 3a,b) and divergent (brown) dynamics during development. The expression of reference promoters was on average higher than those of alternative promoters (Extended Data Fig. 3b). Among 978 transcript pairs with full-length coding sequence annotation, 373 (38%) of the alternative promoters affected only the 5 UTR (for example, dag1; Extended Data Fig. 3c), whereas the remaining 605 altered the N-terminal protein sequence (for example, bmp6; Extended Data Fig. 3d). We analyzed mouse CAGE-seq28 data from comparable embryonic stages and annotated 1,779 multipromoter genes (Extended Data Fig. 3e and Supplementary Table 7). About one-third (294; 30%) of identified mouse orthologs of zebrafish multipromoter genes (974; 75%) utilized alternative promoters. Orthologs of multipromoter genes were likely (P=2.7105; Fishers exact test) to be expressed in similar stages and highly likely (P=3.241058; Fishers exact test) to have multiple promoters in mouse. Multipromoter genes were enriched in KEGG signaling pathways in zebrafish (Fig. 3c) and mouse (Supplementary Table 8), suggesting vertebrate conservation of alternative promoters in signal transduction-associated genes.

Precision promoter annotation and expression dynamics allow exploitation of this resource to predict TF activity regulating the promoters. We implemented Motif Activity Response Analysis (MARA)35,36 for zebrafish. MARA models promoter expression dynamics in terms of the annotated TF binding sites, to infer which TFs most substantially drive expression changes during development. Figure 3d shows the inferred activity profiles of three TFs with strong effects on genome-wide expression patterns. While Tead3 targets are upregulated from gastrulation onwards, Tgif1 targets are transiently downregulated and NF-Y targets are downregulated from the sphere stage onwards, consistent with the known activities of these TFs37,38,39,40,41 (Extended Data Fig. 4 and Supplementary Table 9). MARA predicts substantially changing regulatory activities for regulatory motifs and assigns candidate regulator TFs to promoters (Fig. 3e). We have integrated our zebrafish annotations into the ISMARA webserver (ismara.unibas.ch) to allow this activity analysis on any RNA-seq data.

Next, we aimed to generate a comprehensive atlas of zebrafish developmental regulatory elements. We defined reproducible ATAC-seq42 peaks as PADREs in four pre-zygotic genome activation (ZGA) and seven post-ZGA stages, which we further classified on the basis of the presence of four histone marks using ChromHMM43,44 in five post-ZGA stages (Fig. 4a, Supplementary Fig. 3 and Extended Data Fig. 5a).

a, Genome browser screenshot showing ChromHMM classification of PADREs, and respective histone post-translational modification signals used to define them. b, UMAP plot of PADREs at the Prim-5 stage. Each point represents one open chromatin region, colored by functional assignment. c, Occurrence probabilities of chromatin marks for ChromHMM states. The states function was manually assigned using The Roadmap Epigenomic annotations as reference. 1_TssA1, 2_TssA2: active TSS; 3_TssFlank1, 4_TssFlank2, TSS flanking region; 5_EnhA1, active enhancer; 6_EnhFlank, enhancer flanking region; 7_EnhWk1, primed enhancer; 8_Pois, poised elements; 9_PcRep, Polycomb-repressed regions; 10_Quies, quiescent state. dg, UMAP plot showing PADREs overlapping with CAGE promoters (d), CTCF motif (e), eRNA enhancers (f) and transgenically validated enhancers (g). The transgenically validated enhancers are predominantly associated with enhancer-associated chromatin states (Supplementary Table 11). h, UMAP plot showing the mean phastCons score for each PADRE (top right) and overlap with human CNEs (top left). The bottom subpanel shows the distribution of phastCons scores of active enhancers throughout development (left, bars represents interquartile range), as well as the distribution of the phastCons score for PADREs separated by function at the Prim-5 stage. Two-sided Wilcoxon rank sum test was used to calculate P values between promoters and enhancers (P=2.21016) and enhancers and Polycomb-associated elements (P=2.21016). Exons and intergenic regions were added as reference (right) i, Position of cell-type-specific elements on the UMAP plot (top). ATAC, H3K27ac and H3K4me1 signals around the peak summit of cell-type-specific PADREs (bottom).

a, Openness profile of selected SOM classes (4: early; 6: post-ZGA constitutive; 14: late class), and their position density on the UMAP plots of different developmental stages (top). Heat map of signal intensity of ATAC, H3K27ac and H3K4me1 at the Dome and the Prim-5 stages, along with their respective profiles (bottom). b, Position of COPEs, DOPEs and DOPEs marked with H3K27ac in adult tissues on the UMAP plot (left). Profiles of ATAC, H3K27ac and H3K4me1 of COPEs, DOPEs, DOPEs marked in adult tissues, and other constitutive elements throughout development (right).

To examine the developmental dynamics of PADREs, we developed a UMAP-based method (Methods and Extended Data Fig. 6ac) that can identify known functional classes and potentially novel subclasses during development. The UMAP plot of PADREs (Fig. 4b and Extended Data Fig. 6d) separated most ChromHMM-derived functional classes, including promoters from enhancers (Fig. 4c). Near-symmetry around the y axis reflects strand directionality and was most prominent among promoters (Fig. 4d). Two prominent clusters, which stretched upward and downward from the right apex and bear no chromatin marks, are enriched for the CTCF motif with well-positioned flanking nucleosomes45 (Fig. 4e and Supplementary Fig. 4). Enhancer predictions were validated with two independent sets: (1) enhancers with bidirectional enhancer RNA signals46 called from nuclear CAGE; and (2) a manually curated catalog of published enhancers functionally validated in transgenic reporter assays (Supplementary Table 10). Both colocalized with enhancer-classified PADREs on the UMAP (Fig. 4f,g and Extended Data Fig. 5c,d), demonstrating the utility of the method. DNA methylation analysis revealed CG-rich, promoter-associated PADREs persistently hypomethylated across stages, and less CG-dense enhancer-associated PADREs gradually hypermethylated during development before becoming hypomethylated in adult somatic tissue. Dynamically methylated PADREs varied in the onset and degree of hyper/hypomethylation: for example, conserved phylotypic enhancers11 commenced hypomethylation at the Prim-5 stage (Extended Data Fig. 5f).

Next, we assessed the evolutionary conservation of PADREs by overlapping with human conserved noncoding elements (CNEs) and calculating the phastCons score for each PADRE (Fig. 4h, top, and Extended Data Fig. 5b). Early-acting enhancers appear less conserved than those activated later (Fig. 4h, bottom left, and Extended Data Fig. 5e). phastCons scores of enhancers were higher on average than those of promoters (Fig. 4h, bottom right). Poised elements were the most conserved, suggesting that Polycomb-bound enhancers are a specific class critical for differentiation and organogenesis17,47, and contributing to the hourglass model of development48.

To assign cell-type specificity to PADREs we integrated them with Prim-5 single-cell ATAC-seq30 data (Extended Data Fig. 7a). The majority of anatomical annotation overlapped with transgenically confirmed enhancers and PADRE functional annotation (Extended Data Fig. 7b and Supplementary Table 11). UMAP (Fig. 4i, right) revealed remarkable differences between cell types, both within the same tissue and across tissues. PADREs active in neural precursors of the developing central nervous system showed a threefold increase of H3K27ac compared to those active in differentiating neurons, confirming previous observations about heterogeneity of cell-type population and chromatin dynamics in the developing central nervous system49,50. In contrast, PADREs active in muscle cells carried levels of H3K27ac and H3K4me1 comparable to neural precursors, but distinct accessibility profiles (Fig. 4i, bottom).

To understand the temporal dynamics of PADREs, we created a set of consensus PADREs (cPADREs), containing ~140,000 regions open in at least two neighboring stages (Supplementary Fig. 3a). We clustered nonpromoter cPADREs by chromatin accessibility into self-organizing maps (SOMs) (Extended Data Fig. 7c). Figure 5a (top) shows UMAP locations of 3 out of 16 SOM clusters, which demonstrate remarkable developmental chromatin changes, containing cPADREs active early and subsequently decommissioned (class 4), active from ZGA onwards (class 6) and late elements (class 14). Their chromatin profiles around ATAC-seq peaks were different, with only the early elements depleted of H3K27ac at their peak (Fig. 5a, bottom). With distinct chromatin and conservation profiles, early and late elements represent two separate classes of enhancers.

Finally, we explored the dynamics of PADREs without observable chromatin marks at any stage of development. 2,109 such regions were constitutively open throughout development (Supplementary Fig. 5a), which we termed constitutive orphan predicted elements (COPEs). They colocalized with constitutive SOM class 6 and 40% of them contained a CTCF motif (Figs. 5b, top, and 4e). In contrast, another nonmarked open chromatin set (11,044; termed dynamic orphan predicted elements; DOPES) was open only in specific developmental stages (Fig. 5b and Supplementary Fig. 5b). They were depleted of promoters, with only 65 (0.6%) overlapping CAGE promoters (Supplementary Table 12). Using data from ref. 24, we found that 2,513 DOPEs contained active chromatin marks later in adult tissues, but were open to the same extent as active enhancers already in the embryo (Supplementary Fig. 5c). As we are unaware of epigenetically orphaned accessible elements in the development, whose chromatin opening precedes or is uncoupled from enhancer-associated histone mark deposition, this may represent a discovery of a previously unknown subtype of primed enhancers.

To reveal any developmental promoter regulatory principles, we exploited the PADRE chromatin features to functionally classify CAGE-seq-defined active RNA polymerase II (Pol II) promoters. First, we characterized these promoters at Dome and Prim-5 stages on the basis of their chromatin accessibility at nucleosome resolution, revealing eight clusters (Fig. 6a, Supplementary Fig. 6a and Supplementary Table 13). We detected similar clusters in human embryonic stem cells (Supplementary Fig. 6b) indicating conservation of promoter chromatin architecture classes. The classes differed mostly in their upstream configuration, including the width of the nucleosome-free region (NFR), the signal strength of the central NFR and the presence of upstream open regions (Fig. 6a), which followed GC content (Supplementary Fig. 6c). The NFRs only differed in their amplitude between medium constitutive and weak open (Supplementary Fig. 6a), with the latter either reflecting reduced promoter activity or promoters active only in a subset of cells. The NFR variations were characterized by histone marker presence and patterns of upstream opposite strand transcription (for example, upstream offset) with distinct distances between the main TSS and flanking nucleosomes (for example, wide and strong open) and TSS profiles (Supplementary Fig. 6d). These classes showed notable differences in histone modification patterns (Fig. 6b), confirmed by the differing UMAP positions of promoter PADREs (Fig. 6c). Apart from weak open, each class produces antisense transcription (PROMPTs)51,52,53, including double NFR, wide and upstream offset classes, which showed CAGE expression from both the main NFR and another upstream region, with sense transcription being stronger than antisense (Fig. 6a). Notably, the architecture classes remain stable over developmental time (Fig. 6d and Supplementary Fig. 6e), suggesting they represent distinct regulation mechanisms acting on the genes rather than stage-dependent promoter activity states. Wide and strong open classes contained the most conserved promoters (Fig. 6e and Supplementary Fig. 6f), and were enriched in transcription regulator genes (Fig. 6g and Supplementary Fig. 6g). However, promoter classes showed distinct dynamic temporal expression (Fig. 6f) with notable enrichment of the double NFR class for maternally expressed genes, in contrast to the predominantly early and late zygotic weak open and medium zygotic classes, respectively. The promoter classes also showed distinct gene ontology (GO) enrichment categories (Fig. 6g). Overall, our approach offers a promoter architecture classification for zebrafish and indicates functional specialization and vertebrate conservation of promoter classes.

a, Heat map of chromatin accessibility profiles aligned to dominant TSS per promoter at the Prim-5 stage. Nucleosome-free regions (red) are superimposed with nucleosome positioning (blue). Stack height reflects number of promoters. Above each heat map, combined histograms of CAGE expression are shown. Black, forward TSSs; red, reverse orientation TSSs (the scale is amplified 10 in relation to forward transcription). Nucleosome positioning is symbolized above alignments and black arrows indicate transcription direction; size indicates relative strength. Promoter configuration classes are color-coded consistently in all panels (including Supplementary Fig. 6) b, Aggregated H3K4me1, H3K4me3 (MNase-digested), H3K27ac ChIPseq signals for classes as in a are aligned to dominant TSS. c, UMAP profiles of promoter classes at the Prim-5 stage. UMAPs are cropped to highlight promoter PADREs. d, Flow diagram indicates the relationship between promoter configuration class at the Dome stage (left edge, Supplementary Fig. 6) and the Prim-5 stage (right edge). Band width represents the number of promoters. e, Violin plot of phastCons vertebrate conservation distribution of promoters. Each class is aligned to a. f, Classification of promoter expression during development with SOMs. On the top right, 55 diagrams contain violin plots with stage-by-stage expression levels. Blue to red spectrum indicates maternal to zygotic expression dynamics of promoter clusters. Surface areas of gray circles indicate the number of promoters per cluster. Stages of development are symbolized below the SOM array. On the left, mustard: positive and green: negative color spectrum in SOMs indicates the enrichment in promoter overlap between promoter expression classes (SOMs) in each chromatin architecture class a. g, Enriched GO categories for each promoter architecture class.

Key genes regulating development are controlled by numerous long-range enhancers, which often overlap with highly conserved noncoding elements (HCNEs) within genomic regulatory blocks (GRBs)15, which also often contain other bystander genes that do not respond to those enhancers. The extent of GRBs coincides with those of topologically associating domains (TADs) around developmental genes54 (Fig. 7a). We exploited DANIO-CODE annotations to characterize chromatin opening and interaction topology in those poorly understood loci, and their regulatory role in TADs.

a, Schematic representation of GRBs. Basic components of a GRB. GRB enhancers (green) regulating the target genes span the entire length of the GRB (middle). Typical density pattern of conserved noncoding elements in a GRB, most of which overlap enhancers (top). Hi-C contact matrix within a GRB (bottom). b, Chromatin opening profiles through developmental stages along TADs. c, Genome browser view of a GRB TAD showing H3K27ac signals in the Dome and the Prim-5 stages, H3K27ac ensembles (black bar), CAGE promoters (black blocs) and nonpromoter PADREs (blue active in the Dome stage, red active in the Prim-5 stage and purple PADREs active in both stages). A zoomed-in genome browser view of an H3K27ac ensemble (top, left). d, Aggregate contact enrichment centered on ensembles at stages as indicated. e, TAD compartment score distribution. Positive scores represent A compartments, while negative ones represent B compartments. The comparison was done using two-sided two-sample unpaired Wilcoxon test. g, Heat maps of H3K27ac signal across GRB TADs containing ensembles through developmental stages. TADs are ordered by their width in descending order and fixed on the TAD center. h, CAGE expression patterns of selected gene classes separated by SOM, with the highest and lowest ratios in ensemble-associated genes. Bar plot on the right shows the proportion of ensemble-associated genes in each class. BGT and GST classes are marked on the heat map i, Gene expression pattern of GRB target and bystander genes. The left side bar shows an ensemble association for each gene. The right side bar shows the target or bystander assignment for each gene. Genes in TADs with and without ensembles are separated by a green line. BST and GST classes are indicated on the side. j, Graph showing mean expression and standard error of GRB target genes associated and not associated with early H3K27ac ensembles. k, A model describing the influence of an H3K27ac ensemble on expression of GRB target genes. If the H3K27ac ensemble is in contact with the target gene, it can be expressed early on.

We distinguished GRB TADs, characterized by a high density of extreme noncoding conservation, from non-GRB TADs. In the regions corresponding to late (Long-pec) embryo TADs, chromatin started opening at the boundaries as early as the Dome stage and remains open thereafter (Fig. 7b and Extended Data Fig. 8a). GRB TADs showed a strong increase in accessibility across the entire TAD, whereas in non-GRB TADs the increase was mild and occurred later (Fig. 7b). TADs started to form early but formed fully only at later developmental stages55,56 (Extended Data Fig. 8b). We found more promoter-proximal enhancers in early stages and more distal enhancers in late stages, (Extended Data Fig. 8c), in line with similar findings by contact analysis55.

When we estimated the activity of enhancer candidates by H3K27ac in TADs, we observed that such elements in late stages are numerous, short and distributed throughout the entire TAD length. In contrast, many fewer PADREs were active early at Dome stage, and they often occurred in clusters with uninterrupted H3K27ac signal connecting them (Extended Data Fig. 8d,e and Fig. 7c). We detected ~1,600 such clusters57, of which ~1,300 fell in TADs and were enriched in GRB TADs (Extended Data Fig. 8f). These clusters were reminiscent of super-enhancers57,58, although more numerous than 231 reported in mouse57 and 411 in zebrafish56. Given their unusual scale and early appearance before lineage determination (when previously reported super-enhancers appear), we distinguished them from super-enhancers and called them H3K27ac ensembles. We hypothesized that they might be associated with the lack of fully formed TADs in the early stages, when enhancers are used proximally to early active promoters. To test this, we investigated the relationship between the chromatin interactions and activity of H3K27ac ensemble-associated genes during early versus late embryogenesis.

We found that promoters were enriched at the boundaries of H3K27ac ensembles (Extended Data Fig. 8g) and that the ensembles contain most candidate enhancer PADREs detected in early stages (Extended Data Fig. 8h). In contrast, the PADREs active only later in development represented long-range enhancers, distributed across the entire TAD (Extended Data Fig. 8d), and not enriched in ensembles (Extended Data Fig. 8h). Moreover, H3K27ac present along the entire length of the ensemble became restricted to individual peaks associated with PADREs by Prim-5 (Fig. 7c, zoomed-in panel).

Consistent with an H3K27ac ensemble role in early gene regulation, we observed increased Hi-C contacts within them at Dome in both GRB and non-GRB TADs. By Prim-5, strong contacts spread throughout the entire TAD (Fig. 7d and Extended Data Fig. 9a). TADs with H3K27ac ensembles present at Dome belonged to the active A compartment at Prim-5 (Fig. 7e), arguing for a role for H3K27ac ensembles in the timely opening of chromatin in their host TADs. Indeed, in GRB TADs, the H3K27ac mark propagated from H3K27ac ensembles to fill the entire TAD in later stages (Fig. 7f).

To examine how H3K27ac ensembles influence gene expression, we classified promoters within TADs by expression dynamics using SOM (Extended Data Fig. 9b). H3K27ac ensemble-associated promoters mostly sequestered into clusters, with the highest expression in early post-ZGA stages (Fig. 7g). We termed the top two H3K27ac ensemble-associated classes as blastula-gastrula transition (BGT) and gastrula-segmentation transition (GST) on the basis of the peak expression time. The two major gene classes in GRB TADs were ubiquitously expressed (GRB bystanders) and late zygotic expressed (likely GRB target genes). However, in GRB TADs with an ensemble, we observed a BGT gene class, not present in GRB TADs without an ensemble, as well as more genes in the GST class. Both classes were enriched in ensemble-associated genes (Fig. 7h). Moreover, there was a clear trend of earlier expression in H3K27ac ensemble-associated GRB target genes, compared to other GRB target genes (Fig. 7i), suggesting that ensembles participated in the activation of early-acting developmental genes, including those later dependent on long-range regulation. Moreover, if the target gene is not in contact with the H3K27ac ensemble, it can only become expressed once long-range interactions are present (Fig. 7j).

Next, we investigated whether our annotation of noncoding elements could be exploited to predict functionally conserved cis-regulatory elements (CREs) among vertebrates. Existing comparative methods rely on direct alignments between species of interest59,60. However, the large evolutionary distance between fish and mammals limits the power of comparison, due to loss of noncoding sequence similarity. We developed a method to predict functional conservation across large evolutionary distances and genomic scales independent of direct sequence alignment, exploiting the fact that functional elements often maintain collinear syntenic positions, while their spacing scales with genome size, particularly in GRB TADs15,54,61,62. We selected 13 high-quality bridging species reference genomes and using stepped pairwise sequence alignment (Extended Data Fig. 10 and Methods), which allowed us to map coordinates between genomes of varying sizes, identified reference points (multispecies anchors; Fig. 8a) between genomes and enabling identification of syntenic regions through interpolation of relative syntenic positions between anchor points.

a, Cross-species comparison of the irx3/5(a) TAD between zebrafish and mouse and a zoom-in on the locus around irx3(a). Connecting lines represent projections of bin centers from zebrafish to mouse. b, Distribution of distances from the bin centers (n=528,830) to their closest anchors in zebrafish (blue), and from their projections to their closest anchors in mouse (red), using the direct and the multispecies projection approach. c, Epigenetic comparison of the irx3/5(a) TAD. H3K27me3 overlap in mapped regions is indicated as colored bars (yellow, mutually enriched; blue, zebrafish specific; red, mouse specific; Methods). Opacity reflects signal amplitude and is proportional to the maximum H3K27me3 signal in both species. d, H3K27me3 overlap profiles for four selected GRB TADs. TAD boundaries are indicated with square brackets. e, H3K27me3 overlap profiles of all GRB TADs. TADs are ordered by their relative amount of shared signal. Bins are ordered by the amount of shared signal: bins with shared signal appear in the middle, bins with zebrafish- and mouse-specific signals are left and right, respectively. A view of the TADs with their genomic bin order is given in Extended Data Fig. 10d,f. Classification of zebrafish ATAC-seq peaks in the irx3a locus into DC, IC and NC on the basis of overlaps with direct anchors, multispecies anchors and mouse DNase-seq peak projections (Methods). g, Distribution of DNase-seq signal in the mouse genome around the projected regions of the zebrafish ATAC-seq peaks (n=140,633). Asterisks above the bars indicate the effect size category based on Cohens d: very small (not indicated), small (*), medium (**), large (***), very large (****). h, Cross-species comparison of ChromHMM functional states. i, Cumulative distribution of shared motifs in mouse DNase-seq peaks overlapping zebrafish ATAC-seq peaks. j, H3K27ac enrichment (signal 80th percentile) within (n=11,083) and outside (n=93,020) of enhancer ensembles (P<2.21016, Fishers exact test). k, Cross-species comparison of H3K27ac profile around an H3K27ac ensemble neighboring the zebrafish aktip gene.

We then compared zebrafish and mouse GRB TADs, which differ in size approximately twofold (Extended Data Fig. 10a). We defined GRB TADs as the 1,000 TADs with the highest CNE density, split them into 1-kilobase (kb) bins, and mapped the bin centers from zebrafish to mouse. Using our multispecies approach over direct alignment reduced distances from the bin centers to their closest anchor by a factor of 16 in zebrafish and 29 in mouse (Fig. 8b).

We asked whether this method could discover conserved epigenomic subdomains by comparing epigenomic feature distribution across genomes. We used H3K27me3 ChIPseq data from phylotypic stages in zebrafish (Prim-5) and mouse (E10.5; Methods). H3K27me3 coordinates from zebrafish were projected onto the mouse genome, recovering mouse H3K27me3 features in the corresponding region. An example at the irx3a locus (Fig. 8c) shows H3K27me3 enrichment correlates between zebrafish and mouse, even in the absence of direct sequence conservation. On a genome-wide level, H3K27me3 enrichment is substantially more likely to be shared between zebrafish and mouse for both directly alignable and nonalignable genomic regions (Extended Data Fig. 10e), suggesting epigenomic subdomains and functional elements can be conserved in location and span. We see more GRB TADs showing regions of strong similarity in H3K27me3 extent, while others, such as TADs containing her9 or celf5a, show more zebrafish- or mouse-specific signal enrichment, and still others show little enrichment (Fig. 8d,e).

We next looked at conservation of functional elements marked by open chromatin. We classified zebrafish ATAC-seq peaks in the GRB TADs as directly conserved (DC) if they fall in a region of direct sequence alignment with mouse (16,188 elements, 11.5 %), indirectly conserved (IC) if they do not directly align (6,137 elements, 4.4%) but were alignable through bridging species and nonconserved (NC) for all other peaks (for example, irx3a in Fig. 8f). Notably, DC and IC elements shared regulatory features with their matched counterparts in mouse, including DNase hypersensitivity and ChromHMM feature classification, compared to NC elements (Fig. 8g,h). DC and IC regions were also more likely to share TF binding site (TFBS) motifs compared to nonoverlapping, randomly sampled mouse DNase-seq peaks within and across TAD boundaries (cis and trans in Fig. 8i and Supplementary Table 14). These results suggest a similar level of functional conservation of DC and IC elements, even though IC elements lack direct alignability. Next, we tested whether the early developmental H3K27ac ensembles detected in zebrafish embryos (Fig. 7) are conserved in mouse using our anchoring-based approach. As shown in Fig. 8j,k, H3K27ac signal in mouse was substantially enriched in zebrafish ensembles, suggesting these ensembles are evolutionarily conserved epigenetic subdomains in vertebrates. Genes associated with these conserved ensembles are listed in Supplementary Table 15. Our comparative epigenomic approach has maximized the identification of putative functional elements and epigenetic subdomains conserved between zebrafish and mouse, and highlights the utility of the DANIO-CODE annotations for discovery of vertebrate-conserved mechanisms.

Read more from the original source:
Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements - Nature.com