3796 PART 20 Frontiers

of NF-κB-stimulated inflammatory genes to prevent activation during

asthma treatment.

Type 1 IFN responses are exceptional examples of regulatory complexity governed by epigenetic control. In an unstimulated state, the

H3K9 methyltransferases G9a (EHMT2) and EHMT1 suppress expression

of IFN and IFN-induced genes. Upon induction of IFN-stimulated

genes, STAT transcription factors recruit chromatin remodeling complexes, such as BAF (SMARCA4), and recruit HATs including p300,

CBP, and GCN5 (KAT2A). In turn, chromatin remodeling and acetylation recruit chromatin binding proteins including the bromodomain

protein, BRD4, which promotes transcriptional elongation and full

activation. Beyond the DNA level, METTL3-mediated m6

A methylation on mRNAs also is a critical regulator of IFN signaling in a variety

of distinct cellular contexts.

Major regulators of adaptive immunity pathways are similarly

epigenetically regulated. CD4+ and CD8+ T cells undergo extensive

changes in histone modification profiles during differentiation to

distinct subsets of effector T cells. For example, genes associated with

effector T-cell functions in CD8+ memory T cells (such as PRDM1,

KLRG1, IFNG) display enrichment of H3K4me3 and low levels of

H3K27me3 compared with those genes in naïve T cells. DNA methylation also plays an important regulatory role and may contribute to

disease. For example, CD4+ T cells from individuals with rheumatoid

arthritis (RA), systemic scleroderma, and latent autoimmune diabetes

in adults display hypermethylation of the FOXP3 gene, which activates

regulatory T cells that dampen immune responses. In addition, hypermethylation of the CTLA4 locus occurs in regulatory T cells from RA

patients, impairing their immunosuppressive abilities.

During infection, epigenetic processes can play critical roles in both

the immune response and defense against pathogens, as well strategies

exploited by microorganisms to co-opt the host cellular machinery to

advantage of the pathogen. Respiratory syncytial virus (RSV) infection

promotes the expression of the histone demethylase KDM5B, which

removes H3K4 methyl groups from antiviral genes such as type I IFNs,

driving a switch from T helper 1 to T helper 2–type immune responses,

thereby contributing to chronic infection. Similarly, influenza upregulates the repressive H3K9me3 methyltransferase SETDB2 to block

expression of CXCL1 and a variety of NF-κB target genes involved in

attracting neutrophils and host defense, both serving to lengthen the

infection and contributing to bacterial superinfection. Regarding the

host response to infection, studies have revealed that differences in

host tissue-, age-, and sex-biased epigenetic profiles might shape susceptibility and responses to infection. For example, differential DNA

methylation at the ACE2 gene may impact expression levels of this key

cellular receptor and ultimately the ability of SARS-CoV-2 to infect

hosts, while alterations in antiviral IFN signaling may lead to more

severe COVID-19 infection and disease.

Although mechanistic studies remain limited, there are numerous

examples of epigenetic-based therapies associated with extensive effects

on the immune system, underscoring the potential hope for eventual

treatment of immune-related conditions. For example, the DNA methylation inhibitors azacitidine and decitabine have immunosuppressive

effects possibly mediated by enhanced expression of FOXP3, which

generally suppresses immune responses. HDAC inhibitors upregulate

and downregulate immune genes, and they inhibit cytokine production

in macrophages from patients with RA. Further, the HDAC inhibitors

vorinostat and panobinostat inhibit primary B-cell responses and

antibody production in vitro and in vivo. Given these broad effects,

it is not surprising that the HDAC inhibitor trichostatin A (TSA) has

efficacy in various model systems for treatment of RA, systemic lupus

erythematosus (SLE), asthma, acute kidney injury, sepsis-induced

lung and cardiac damage, and acute pancreatitis. Similarly, BETi also

display broad effects in blocking antigen presentation and T- and

B-cell activation and thus beneficial protective effects in a variety of

inflammatory settings including autoimmunity, sepsis, atherosclerosis,

psoriasis, periodontitis, and arthritis. Beyond these “broad-spectrum”

epigenetic inhibitors, GSK-J4, which is a specific inhibitor of the

H3K27me3 demethylases KDM6A and KDM6B, has anti-inflammatory

activity, presumably by preventing loss of H3K27me3 repression over

inflammatory genes. Similarly, inhibition of the H3K4me3 histone

methyltransferase, MLL1 blocks the induction of proinflammatory

cytokine gene expression in a variety of contexts.

CONCLUSIONS

Due to the enormity and complexity of the chromatin and epigenetics

fields and their reach into all areas of biology and medicine, it is not

possible to cover such a broad scope in a single chapter. Thus, here we

provide a concise snapshot highlighting key areas of development in

medicine. We hope to have conveyed the tremendous excitement and

promise that pervades the discipline. Indeed, given the exponential

growth in uncovering the interface between the epigenome and epigenetic therapies with the environment and disease, there is little doubt

that the coming years will bring important additions to this field.

■ FURTHER READING

Allis CD, Jenuwein T: The molecular hallmarks of epigenetic control.

Nat Rev Genet 17:487, 2016.

Avgistinova A, Benitah SA: Epigenetic control of adult stem cell

function. Nat Rev Mol Cell Biol 17:643, 2016.

Cavalli G, Heard E: Advances in epigenetics link genetics to environment and disease. Nature 571:489, 2019.

Dai Z et al: The evolving metabolic landscape of chromatin biology

and epigenetics. Nat Rev Genet 21:737, 2020.

Hwang JY et al: The emerging field of epigenetics in neurodegeneration and neuroprotection. Nat Rev Neurosci 18:347, 2017.

Mohammad HP et al: Targeting epigenetic modifications in cancer

therapy: Erasing the roadmap to cancer. Nat Med 25:403, 2019.

Tough DF et al: Epigenetic drug discovery: Breaking through the

immune barrier. Nat Rev Drug Discov 15:835, 2016.

Zaccara S et al: Reading, writing and erasing mRNA methylation. Nat

Rev Mol Cell Biol 20:608, 2019.

Zhang Q et al: Epigenetic regulation of the innate immune response to

infection. Nat Rev Immunol 19:417, 2019.

Zhang W et al: The ageing epigenome and its rejuvenation. Nat Rev

Mol Cell Biol 21:137, 2020.

Damage to an organ initiates a series of events that lead to the reconstruction of the damaged tissue, including proliferation, differentiation,

and migration of various cell types; release of cytokines and chemokines; and remodeling of the extracellular matrix. Endogenous stem and

progenitor cells are among the cell populations that are involved in these

injury responses. In normal steady-state conditions, an equilibrium

is maintained in which endogenous stem cells intrinsic to the tissue

replenish dying cells. After tissue injury, stem cells in organs such as the

liver and skin have a remarkable ability to regenerate the organ, whereas

other stem cell populations, such as those in the heart and brain, have

a much more limited capability for self-repair. In rare circumstances,

circulating stem cells may contribute to regenerative responses by

migrating into a tissue and differentiating into organ-specific cell types.

The goal of stem cell therapies is to promote cell replacement in organs

that are damaged beyond their ability to self-repair.

■ GENERAL STRATEGIES FOR STEM CELL

REPLACEMENT

At least three different therapeutic concepts for cell replacement can

be envisaged (Fig. 484-1). One therapeutic approach involves direct

484 Applications of

Stem Cell Biology in

Clinical Medicine

John A. Kessler

3797Applications of Stem Cell Biology in Clinical Medicine CHAPTER 484

are often used to replace damaged tissues. However, the need for transplantable tissues and

organs far outweighs the available supply, and

organ transplantation has limited potential for

some tissues, such as the brain. Stem cells offer

the possibility of a renewable source of replacement cells for virtually all organs.

■ SOURCES OF STEM CELLS FOR

TISSUE REPAIR

A variety of different types of stem cells could

be used in regenerative strategies, including

embryonic stem (ES) cells, induced pluripotent

stem (iPS) cells, umbilical-cord blood stem cells

(USCs), organ-specific somatic stem cells (e.g.,

neural stem cells for treatment of the brain),

and somatic stem cells that generate cell types

specific for the target organ rather than the

donor organ (e.g., bone marrow mesenchymal

stem cells [MSCs] or CD34+ HSCs for cardiac

repair). Although each cell type has potential advantages and disadvantages, there are a

number of generic challenges associated with

developing any of these cell types into a useful

and reliable clinical tool.

Embryonic Stem Cells ES cells have the

potential to generate all of the cell types in the

body; thus, in theory, there are no restrictions

on the organs that could be regenerated. ES cells

can self-renew endlessly, so that a single cell line

with carefully characterized traits potentially

could generate almost limitless numbers of

cells. In the absence of moral or ethical constraints (see “Ethical Issues,” below), unused

human blastocysts from fertility clinics could be used to derive new ES

cell lines that are matched immunologically with potential transplant

recipients. ES cells also can be derived from unfertilized oocytes by

parthenogenesis, but there is debate about whether this alters the ethical issues. However, human ES cells are difficult to culture and grow

slowly. Techniques for differentiating them into specific cell types are

still nascent. Cells tend to develop abnormal karyotypes and other

abnormalities with increased time in culture, and ES cells have the

potential to form teratomas if all cells are not committed to the desired

cell types before transplantation. Further, human ES cells are ethically

controversial, and on these grounds, their use would be unacceptable

to some patients and physicians despite their therapeutic potential.

Nevertheless, there have been limited clinical trials of ES-derived cells

in a number of disorders, including macular degeneration, myopia,

heart failure, diabetes, and spinal cord injury (SCI).

Induced Pluripotent Stem Cells The field of stem cell biology

was transformed by the discovery that adult somatic cells can be

converted (“reprogrammed”) into pluripotent cells through the overexpression of four transcription factors normally expressed in pluripotent cells. These iPS cells share most properties with ES cells, although

there are distinct differences in gene expression between ES and iPS

cells. The initial use of viruses to insert the transcription factors into

somatic cells made the resulting cells unsuitable for clinical use. However, a number of strategies have since been developed to circumvent

this problem, including the insertion of modified mRNAs, proteins, or

microRNAs rather than cDNAs and treatment with small molecules;

the use of nonintegrating viruses such as Sendai virus; the insertion

of transposons with the programming factors, followed by their subsequent removal; and the use of floxed viral constructs, followed by

treatment with Cre recombinase to excise those constructs. iPS cells

derived from patients with different disorders are currently being used

extensively for disease modeling and drug discovery. However, the

safety of iPS cells for use in regenerative strategies in humans remains

Undifferentiated

stem cells

Undifferentiated

stem cells

Erythropoietin

Dopaminergic

neurons

Erythrocytes

Hematopoietic

stem cells

Into striatum

Into heart

Intravenous

FIGURE 484-1 Strategies for transplantation of stem cells. 1. Undifferentiated or partially differentiated stem

cells may be injected directly into the target organ or intravenously. 2. Stem cells may be differentiated ex vivo

before injection into the target organ. 3. Growth factors or other drugs may be injected to stimulate endogenous

stem cell populations.

administration of stem cells. The cells may be injected directly into the

damaged organ, where they can differentiate into the desired cell type.

Alternatively, stem cells may be injected systemically since they have

the capacity to home in on damaged tissues by following gradients of

cytokines and chemokines released by the diseased organ. A second

approach involves transplantation of differentiated cells derived from

stem cells. For example, pancreatic islet cells can be generated from

stem cells before transplantation into diabetic patients, and cardiomyocytes can be generated to treat heart disease. A third approach involves

stimulation of endogenous stem cells to facilitate repair. This goal

might be accomplished by administration of appropriate growth factors

and drugs that amplify the number of endogenous stem/progenitor

cells and/or direct them to differentiate into the desired cell types.

Therapeutic stimulation of precursor cells is already a clinical reality in

the hematopoietic system, where factors such as erythropoietin, granulocyte colony-stimulating factor, and granulocyte-macrophage colonystimulating factor are used to increase production of specific blood

elements. In addition to these strategies for cell replacement, a number

of other approaches could involve stem cells for ex vivo or in situ generation of tissues, a process termed tissue engineering. Stem cells are

excellent candidates as vehicles for cellular gene therapy (Chap. 470),

and they also potentially can be used to modify immune responses.

For example, genetically modified T cells have been used treat various

type of malignancies. Administration of components of stem cells, such

as exosomes, also may be used to stimulate regenerative responses.

Finally, transplanted stem cells may exert paracrine effects to promote

repair of damaged tissues without differentiating to replace lost cells.

Stem cell transplantation is not a new concept but rather is already

part of established medical practice. Hematopoietic stem cells (HSCs)

(Chap. 96) are responsible for the long-term repopulation of all blood

elements in recipients of bone marrow transplants, and hematopoietic

stem cell transplantation is the gold standard against which other stem

cell transplantation therapies are measured. Transplantation of differentiated cells is also a clinical reality, and donated organs and tissues

3798 PART 20 Frontiers

to be demonstrated. The first clinical trial in macular degeneration was

initially suspended after treatment of one patient because of discovery

of a mutation in cells derived for the second patient, but the trial was

later resumed and progressed without significant safety issues. In

addition to ongoing clinical trials in macular degeneration, trials of

iPS cells also have commenced for treatment of heart failure, SCI, and

Parkinson’s disease, and trials in numerous other disorders are planned.

An advantage of iPS cells is that somatic cells from patients would

generate pluripotent cells genetically identical to those of the patient,

obviating the need for immunosuppression. Further, these cells are not

subject to the same ethical constraints as ES cells. It is not clear whether

the differences in gene expression between ES and iPS cells will have

any impact on their potential clinical utility, and studies of both cell

types will be needed to resolve this issue.

Umbilical-Cord Stem Cells USCs are widely available. These

cells appear to be associated with less graft-versus-host disease than are

some other cell types, such as marrow stem cells. They have less human

leukocyte antigen restriction than adult marrow stem cells and are less

likely to be contaminated with herpesvirus. However, it is unclear how

many different cell types can be generated from USCs, and methods

for differentiating these cells into nonhematopoietic phenotypes are

largely lacking. Nevertheless, there are ongoing clinical trials of these

cells in dozens of disorders, including cirrhosis, cardiomyopathies,

multiple sclerosis, burns, stroke, autism, and critical limb ischemia, and

cord stem cells have been approved by the Food and Drug Administration for the treatment of certain hematopoietic disorders and cancers.

Organ-Specific Multipotent Stem Cells Organ-specific multipotent stem cells have the advantage of already being somewhat specialized so that the induction of desired cell types may be easier. Cells

potentially could be obtained from the patient and amplified in cell culture, circumventing the problems associated with immune rejection.

Stem cells are relatively easy to harvest from some tissues, such as bone

marrow and blood, but are difficult to harvest from other tissues, such

as heart and brain. Moreover, these populations of cells are more limited in potentiality than are pluripotent ES or iPS cells, and they may be

difficult to obtain in large quantities from many organs. Therefore, substantial efforts have been devoted to developing techniques for using

more easily obtainable stem cell populations, such as bone marrow

MSCs, CD34+ HSCs, cardiac mesenchymal cells, and adipose-derived

stem cells (ASCs), for use in regenerative strategies. Tissue culture evidence suggests that these stem cell populations may be able to generate

differentiated cell types unrelated to their organ source (including

myocytes, chondrocytes, tendon cells, osteoblasts, cardiomyocytes,

adipocytes, hepatocytes, and possibly neurons) in a process known as

transdifferentiation. However, it is still unclear whether these stem cells

are capable of generating differentiated cell types that integrate into

organs, survive, and function after transplantation in vivo. A number

of early studies of MSCs transplanted into heart, liver, and other organs

suggested that the cells had differentiated into organ-specific cell types

with beneficial effects in animal models of disease. Unfortunately,

subsequent studies revealed that the stem cells had simply fused with

cells resident in the organs and that the observed beneficial effects were

due to paracrine release of trophic and anti-inflammatory cytokines.

Further studies will be necessary to determine whether transdifferentiation of MSCs, ASCs, or other stem cell populations occurs at a high

enough frequency to make these cells useful for stem cell replacement

therapy. Despite the remaining issues, a large number of clinical trials

of MSCs, autologous HSCs, USCs, and ASCs are being performed in

many disorders, including ischemic cardiac disease, cardiomyopathy,

diabetes, stroke, cirrhosis, amyotrophic lateral sclerosis (ALS), muscular dystrophy, and other disorders. However, in general, therapeutic

benefits to humans have not been as robust as the findings in animal

models. Another approach has been to derive organ-specific stem cells

by parthenogenesis, and a clinical trial of neural stem cells derived this

way has begun for Parkinson’s disease.

Regardless of the source of the stem cells used in regenerative strategies, a number of generic problems must be overcome for the development of successful clinical applications. Methods must be devised to

reliably generate large numbers of specific cell types, to minimize the

risk of tumor formation or proliferation of inappropriate cell types, to

ensure the viability and function of the engrafted cells, to overcome

immune rejection when autografts are not used, and to facilitate revascularization of regenerated tissue. Each organ system also will pose

tissue-specific problems for stem cell therapies.

■ DISEASE-SPECIFIC APPLICATIONS OF STEM

CELLS

Ischemic Heart Disease and Cardiomyocyte Regeneration

Because of the high prevalence of ischemic heart disease, extensive

efforts have been devoted to the development of strategies for stem

cell replacement of cardiomyocytes. Historically, the adult heart has

been viewed as a terminally differentiated organ without the capacity

for regeneration. However, recent studies have demonstrated that the

heart has the capacity for low levels of cardiomyocyte regeneration

(Chap. 237). This regeneration appears to be accomplished by cardiac

stem cells resident in the heart and possibly also by cells originating

in the bone marrow. The heart might be an ideal source of stem cells

for therapeutic use, but techniques for isolating, characterizing, and

amplifying large numbers of these cells have not yet been perfected. For

successful myocardial repair, stem cell therapy must deliver cells either

systemically or locally, and the cells must survive, engraft, and differentiate into functional cardiomyocytes that couple mechanically and

electrically with the recipient myocardium. The optimal method for

cell delivery is not clear, and various experimental and clinical studies

have employed intramyocardial, transendocardial, intravenous, intracoronary, and retrograde coronary venous injections. In experimental

myocardial infarction, functional improvements have been achieved

after transplantation of a variety of different cell types, including ES

cells, HSCs, MSCs, USCs, and ASCs. Early studies suggested that each

of these cell types might have the potential to engraft and generate cardiomyocytes. However, most investigators have found that the generation of new cardiomyocytes by these cells is at best a rare event and that

graft survival over long periods is poor. The preponderance of evidence

suggests that the observed beneficial effects of most experimental therapies were not derived from direct stem cell generation of cardiomyocytes but rather from indirect effects of the stem cells on resident cells.

It is not clear whether these effects reflect the release of soluble trophic

factors, the induction of angiogenesis, the release of anti-inflammatory

cytokines, or another mechanism. A wide variety of cell delivery methods, cell types, and cell doses have been used in a progressively enlarging series of clinical trials, but the fate of the cells and the mechanisms

by which they alter cardiac function are still open questions. A number

of studies have shown a small but measurable improvement in cardiac

function and, in some cases, reduction in infarct size. Further, some

studies have reported that transplantation of bone marrow–derived

stem cells improved outcome for patients in heart failure. However, in

aggregate, the clinical benefits of stem cell therapy have been small and

inconsistent. Moreover, the available evidence suggests that beneficial

clinical effects, if any, reflect an indirect effect of the transplanted cells

rather than cell replacement. However, genuine cell replacement may

become possible as new protocols are being developed for generating

cardiomyocytes from pluripotent and multipotent stem cells.

Diabetes Successes with islet cell and pancreas transplantation have

provided proof of concept for cell-based therapies for type 1 diabetes.

However, the demand for donor pancreases far exceeds the number

available, and maintenance of long-term graft survival remains a problem. The search for a renewable source of stem cells capable of regenerating pancreatic islets has therefore been intensive. Pancreatic beta

cell turnover occurs even in the normal pancreas, although the source

of the new beta cells remains controversial. This persistent turnover

suggests that, in principle, it should be possible to develop strategies

for reconstituting the beta cell population in diabetics. Attempts to

devise techniques for promoting endogenous regenerative processes

by using combinations of growth factors, drugs, and gene therapy

have failed thus far, but this remains a potentially viable approach.

3799Applications of Stem Cell Biology in Clinical Medicine CHAPTER 484

A number of different cell types are candidates for use in stem cell

replacement strategies, including iPS cells, ES cells, hepatic progenitor

cells, pancreatic ductal progenitor cells, and MSCs. Successful therapy

will depend on the development of a source of cells that can be amplified to produce large numbers of progeny with the ability to synthesize,

store, and release insulin when it is required, primarily in response

to changes in the ambient level of glucose. The proliferative capacity

of the replacement cells must be tightly regulated to avoid excessive

expansion of beta cell numbers and the consequent development of

hyperinsulinemia/hypoglycemia; moreover, the cells must withstand

immune rejection. Several strategies are being examined for preventing

immune rejection including encapsulation of the cells, elimination of

HLA genes, and expression of checkpoint inhibitors. ES and iPS cells

can be differentiated into cells that produce insulin, and implants of

these cells can normalize blood glucose levels in diabetic animals.

Clinical trials of encapsulated ES cell–derived pancreatic progenitor

cells are currently in progress.

During embryogenesis, the pancreas, liver, and gastrointestinal tract

are all derived from the anterior endoderm, and transdifferentiation

of pancreas to liver and vice versa has been observed in a number of

pathologic conditions. There is also substantial evidence that multipotential stem cells reside within gastric glands and intestinal crypts.

These observations suggest that hepatic, pancreatic, and/or gastrointestinal precursor cells may be reasonable candidates for cell-based therapy for diabetes, although it is unclear whether insulin-producing cells

derived from pancreatic stem cells or liver progenitors can be expanded

in vitro to clinically useful numbers. MSCs and neural stem cells both

reportedly have the capacity to generate insulin-producing cells, but

there is no convincing evidence that either cell type will be clinically

useful. Clinical trials of MSCs, USCs, HSCs, and ASCs in both type 1

and type 2 diabetes are ongoing.

Nervous System Substantial progress has been made in the development of methodologies for generating neural cells from different

stem cell populations. Human ES or iPS cells can be induced to generate cells with the properties of neural stem cells, and these cells in

turn give rise to neurons, oligodendroglia, and astrocytes. Reasonably

large numbers of these cells can be transplanted into the rodent brain

with formation of appropriate cell types and no tumor formation.

Multipotent stem cells present in the adult brain also can be easily

amplified in number and used to generate all the major neural cell

types, but the need for invasive procedures to obtain autologous cells

is a major limitation. Fetal neural stem cells derived from miscarriages

or abortions are an alternative but raise ethical concerns. Nevertheless,

clinical trials of fetal neural stem cells have commenced in ALS, stroke,

and several other disorders. Transdifferentiation of MSCs and ASCs

into neural stem cells, and vice versa, has been reported by numerous

investigators, and clinical trials of such cells have begun for a number

of neurologic diseases. Clinical trials of a conditionally immortalized

human cell line and of USCs in stroke are also in progress. Because of

the incapacitating nature of neural disorders and the limited endogenous repair capacity of the nervous system, clinical trials of stem cells

in neurologic disorders have been particularly numerous, including

trials in SCI, multiple sclerosis, epilepsy, Alzheimer’s disease, ALS,

acute and chronic stroke, numerous genetic disorders, traumatic

brain injury, Parkinson’s disease, and others. In diseases such as ALS,

possible benefits are more likely to be due to indirect trophic effects

than to neuron replacement. In Parkinson’s disease, the major motor

features of the disorder result from the loss of a single cell population:

dopaminergic neurons within the substantia nigra; this circumstance

suggests that cell replacement should be relatively straightforward.

However, two clinical trials of fetal nigral transplantation failed to meet

their primary endpoint and were complicated by the development of

dyskinesia. Transplantation of stem cell–derived dopamine-producing

cells offers a number of potential advantages over the fetal transplants,

including the ability of stem cells to migrate and disperse within tissue,

the potential for engineering regulatable release of dopamine, and the

ability to engineer cells to produce factors that will enhance cell survival. Clinical trials of iPSC- and ESC-derived dopaminergic neuron

precursors are in progress, but the experiences with fetal transplants

point out the difficulties that may be encountered.

At least some of the neurologic dysfunction after SCI reflects demyelination, and both ES cells and MSCs can facilitate remyelination

after experimental SCI. Clinical trials of MSCs in this disorder have

commenced in a number of countries, and SCI was the first disorder

targeted for the clinical use of ES cells. The first trial of ES cell–derived

oligodendroglial progenitor cells in SCI was terminated early for

nonmedical reasons, but another trial has commenced. At present,

no population of transplanted stem cells has been shown to have the

capacity to generate neurons that extend axons over long distances to

form synaptic connections (as would be necessary for replacement of

upper motor neurons in ALS, stroke, or other disorders). For many

injuries, including SCI, the balance between scar formation and tissue

repair/regeneration may prove to be an important consideration. For

example, it may ultimately prove necessary to limit scar formation so

that axons can reestablish connections.

Liver Liver transplantation is currently the only successful treatment for end-stage liver diseases, but the shortage of liver grafts limits

its application. Clinical trials of hepatocyte transplantation demonstrate its potential as a substitute for organ transplantation, but this

approach is limited by the paucity of available cells. Potential sources

of stem cells for regenerative strategies include endogenous liver stem

cells (such as oval cells), ES cells, MSCs, and USCs. Although a series of

studies in humans as well as animals suggested that transplanted MSCs

and HSCs can generate hepatocytes, fusion of the transplanted cells

with endogenous liver cells, giving the erroneous appearance of new

hepatocytes, appears to be the underlying event in most circumstances.

The available evidence suggests that transplanted HSCs and MSCs can

generate hepatocyte-like cells in the liver only at a very low frequency,

but there are beneficial consequences presumably related to indirect

paracrine effects. ES cells can be differentiated into hepatocytes and

transplanted in animal models of liver failure without the formation of

teratomas. Clinical trials are in progress in cirrhosis with numerous cell

types, including MSCs, USCs, HSCs, and ASCs.

Other Organ Systems and the Future The use of stem cells

in regenerative strategies has been studied for many other organ systems and cell types, including skin, eye, cartilage, bone, kidney, lung,

endometrium, vascular endothelium, smooth muscle, and striated

muscle, and clinical trials in these and other organs are ongoing. In

fact, the potential for stem cell regeneration of damaged organs and

tissues is virtually limitless. However, numerous obstacles must be

overcome before stem cell therapies can become a widespread clinical

reality. Only HSCs have been adequately characterized by surface

markers so that they can be unambiguously identified, a prerequisite

for reliable clinical applications. The pathways for differentiating

stem cells into specific cellular phenotypes are largely unknown, and

the ability to control the migration of transplanted cells or predict

the response of the cells to the environment of diseased organs is

presently limited. Some strategies may employ the coadministration

of scaffolding, artificial extracellular matrix, and/or growth factors

to orchestrate differentiation of stem cells and their organization into

appropriate constituents of the organ. There is currently no way to

image stem cells in vivo after transplantation into humans, and it will

be necessary to develop techniques to do so. Fortunately, stem cells can

be engineered before transplantation to contain a contrast agent that

may make in vivo imaging feasible. The potential for tumor formation

and the problems associated with immune rejection are impediments,

and it will also be necessary to develop techniques for ensuring vascularization of regenerated tissues. There already are many strategies for

supporting cell replacement, including coadministration of vasoactive

endothelial growth factor to foster vascularization of the transplant.

Some strategies also include the genetic engineering of stem cells with

an inducible suicide gene so that the cells can be easily eradicated in

the event of tumor formation or another complication. The potential

for stem cell therapies to revolutionize medical care is extraordinary,

and disorders such as myocardial infarction, diabetes, and Parkinson’s

disease, among many others, are potentially curable by such therapies.

3800 PART 20 Frontiers

However, stem cell–based therapies are still at a very early stage of

development, and perfection of techniques for clinical transplantation

of predictable, well-characterized cells is going to be a difficult and

lengthy undertaking.

■ ETHICAL ISSUES

Stem cell therapies raise ethical and socially contentious issues that

must be addressed in parallel with the scientific and medical opportunities. Society has great diversity with respect to religious beliefs,

concepts of individual rights, tolerance for uncertainty and risk, and

boundaries for how scientific interventions should be used to alter

the outcome of disease. In the United States, the federal government

has authorized research using existing human ES cell lines but still

restricts the use of federal funds for developing new human ES cell

lines. Ongoing studies of existing lines have indicated that they develop

abnormalities with time in culture and that they may be contaminated

with mouse proteins. These findings highlight the need to develop new

human ES cell lines. As noted above, ES cells may be derived from

unfertilized oocytes by parthenogenesis, but there is debate about

whether this alters the ethical issues. The development of iPS cell technology may lessen the need for deriving new ES cell lines, but it is still

not clear whether the differences in gene expression by ES and iPS cells

are important for potential clinical use.

In considering ethical issues associated with the use of stem cells, it

is helpful to draw from experience with other scientific advances, such

as organ transplantation, recombinant DNA technology, implantation

of mechanical devices, neuroscience and cognitive research, in vitro

fertilization, and prenatal genetic testing. These and other precedents

have pointed to the importance of understanding and testing fundamental biology in the laboratory setting and in animal models before

applying new techniques in carefully controlled clinical trials. When

these trials occur, they must include full informed consent and careful

oversight by external review groups.

Ultimately, there will be medical interventions that are scientifically

feasible but ethically or socially unacceptable to some members of

a society. Stem cell research raises fundamentally difficult questions

about the definition of human life, and it has raised deep fears about

the ability to balance issues of justice and safety with the needs of critically ill patients. Health care providers and experts with backgrounds

in ethics, law, and sociology must help guard against the premature or

inappropriate application of stem cell therapies and the inappropriate

involvement of vulnerable population groups. However, these therapies

offer important new strategies for the treatment of otherwise irreversible disorders. An open dialogue among the scientific community, physicians, patients and their advocates, lawmakers, and the lay population

is critically important to raise and address important ethical issues and

balance the benefits and risks associated with stem cell transfer.

■ FURTHER READING

Blau HM, Daley GQ: Stem cells in the treatment of disease. N Eng J

Med 380:1748, 2019.

De Luca M et al: Advances in stem cell research and therapeutic development. Nat Cell Biol 21:801, 2019.

He L et al: Heart regeneration by endogenous stem cells and cardiomyocyte proliferation: Controversy, fallacy, and progress. Circulation

142:275, 2020.

International Society for Stem Cell Research: Informed consent standard for stem cell–based interventions offered outside of

formal clinical trials, 2019. Available from https://www.isscr.org/docs/

default-source/policy-documents/isscr-informed-consent-standardsfor-stem-cell-based-interventions.pdf.

Levy O et al: Shattering barriers toward clinically meaningful MSC

therapies. Sci Adv 6:eaba6884, 2020.

Li M et al: Organoids: Preclinical model of human disease. N Engl J

Med 380:569, 2019.

Parmar M et al: The future of stem cell therapies for Parkinson disease. Nat Rev Neuroscif 21:103, 2020.

Yamanaka S: Pluripotent stem cell-based cell therapy-promise and

challenges. Cell Stem Cell 27:523, 2020.

Circadian rhythms are anticipatory, circa 24-h, autonomous cycles

of physiology and behavior. These evolutionarily-conserved rhythms

have evolved at both the cell and tissue level to synchronize organismal

function in anticipation of the 24-h rotation of the Earth. A common

feature of modern “24/7” life is the routine disruption of these endogenous circadian cycles due to the rise in shift work, jet travel across time

zones, exposure to blue light–emitting devices at night, and disrupted

sleep-wake behavior. In-depth characterization of the molecular basis

of circadian disorders has generated novel avenues for research on

how sleep-wake disruption has been associated with aging, metabolic

disease, inflammation, and cancer. This chapter provides an overview

of (1) the basic biology of the circadian system; (2) primary circadian

rhythm and interrelated sleep disorders; and (3) the role of the circadian system in both normal human physiology and disease states. We

also include an overview of how the emerging field of chronobiology

may impact drug action. A glossary of terms used in circadian biology

is summarized in Table 485-1.

■ BASIC EVOLUTION AND STRUCTURE OF THE

CIRCADIAN SYSTEM

Long before the emergence of multicellular life, the Earth’s constant rotation around the sun gave rise to a daily cycle of light and

darkness. At the emergence of the first prototypal gene involved in

biological clock regulation—3.4 billion years ago in photosynthetic

cyanobacteria—the period of Earth’s rotation along its own axis was

only 8 h. The co-occurrence in molecular evolution of the biological

clock and photosynthesis hints at an interrelated and selective advantage of the clock in regulation of energetic processes. Indeed, biological

clocks coordinate oxygenic reactions with periods of sunlight each day,

and perturbation of clock cycles reduces fitness, reproduction, and survival. Additionally, clocks protect photosynthetic organisms from the

DNA-damaging effects of sunlight by timing the production of DNA

repair processes, such as photolyase-mediated repair, to the nighttime. Across billions of years of evolution, as day length has gradually

extended to today’s circa 24 h, highly conserved circadian clocks (from

circa diem, meaning “about a day”) have been found in all photosensitive organisms, governing a wide range of biochemical, physiologic,

and behavioral processes. A defining property of the circadian clock

system is that it enables organisms to anticipate, rather than simply

react to, daily changes in the external environment that are tied to the

day-night cycle. In mammals, circadian systems are organized hierarchically with a light-responsive “master” circadian pacemaker located

within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, which in turn presides over a network of both extra-SCN and

peripheral clocks (see “Anatomic Organization of the Circadian Clock

Network,” below). Daily light exposure signals to the SCN and entrains

the circadian system to the 24-h day (see “Entrainment and Measurement of the Circadian System,” below). In turn, the SCN maintains synchrony of a diverse network of both central and peripheral clocks via

a variety of signals that have as yet to be fully identified. These signals

involve direct physiologic rhythms (core body temperature), the autonomic nervous system, and neuroendocrine signals, including cortisol

as part of the hypothalamic-pituitary-adrenal (HPA) axis.

■ MOLECULAR ORGANIZATION OF THE

MAMMALIAN CIRCADIAN CLOCK

At the molecular level, mammalian circadian rhythms are generated by

a transcription-translation autoregulatory feedback loop. The forward

485 The Role of Circadian

Biology in Health

and Disease

Jonathan Cedernaes, Kathryn Moynihan

Ramsey, Joseph Bass

3801The Role of Circadian Biology in Health and Disease CHAPTER 485

TABLE 485-1 Glossary of Terms Used in Discussion of the Circadian System

TERM DESCRIPTION

ASPD Advanced sleep phase disorder (see text for description).

CBT Core body temperature. Often used as an indicator of the circadian rhythm, but can be masked by sleep and exercise.

CCGs Clock-controlled genes; output of the molecular clock.

Chronotype Internal circadian rhythm of an individual determined by phase of entrainment, determining sleep propensity and timing of maximum

alertness over a 24-h period.

Circadian period Time required for one complete cycle or oscillation. Calculated by the time distance between two consecutive peaks or troughs of a

circadian variable.

Circadian phase Timing of the circadian rhythm. Defined by comparing, e.g., the peak (acrophase) or trough (bathyphase) to a fixed event, e.g., to a point

in time. Synonymous with phase angle.

Circadian rhythm A biological process that exhibits an endogenous, entrainable oscillation of ~24 h.

Circadian rhythm sleep

disorders

Disorders of multiple etiology that have in common that they result in maladjustment of the biological clock with respect to the

environment.

Constant routine An experimental paradigm designed to study endogenous circadian rhythms in humans, by keeping behavioral and environmental factors

constant. These paradigms thereby typically entail a combination of constant dim lighting, evenly distributed isocaloric energy intake,

semirecumbent posture, and forced extended wakefulness.

Desynchrony Loss of synchrony occurring either between a rhythm and its zeitgeber (external, “time giver” signal) or between two or more rhythms

within an organism (internal).

Diurnal rhythm An oscillation synchronized with the day/night cycle that repeats itself with a 24-h period. The rhythm does not have to persist when time

cues (e.g., light) are absent.

DLMO Dim-light melatonin onset; a marker of melatonin rhythm.

DSPD Delayed sleep phase disorder (see text for description).

Entrainment Synchronization of a circadian rhythm or other self-sustaining oscillation by a factor—zeitgeber—that enforces the oscillator. Constant

entrainment between the zeitgeber and the oscillator results in a stable phase relationship between these entities.

Infradian rhythm A recurrent cycle or period with a period length significantly greater than 24 h.

Melatonin Hormone produced by the pineal gland (chemical name N-acetyl-5-methoxytryptamine); derived from L-tryptophan. Various forms of

melatonin can be prescribed for circadian rhythm sleep disorders or sleep disorders.

Non-24-h rhythm disorder A syndrome in which there typically are chronic 1- to 2-h daily delays in sleep onset and wake times in an individual living in society,

e.g., due to complete blindness.

Peripheral clocks Clocks presiding outside of the suprachiasmatic nucleus, the circadian system’s master pacemaker.

PRC Phase response curve; visual representation of how a particular manipulation (e.g., light) produces phase shifts as a function of the

phase (i.e., circadian time) at which the manipulation occurs. Defining the PRC to light has enabled researchers to understand and

predict how entrainment to light cycles is accomplished.

SCN The suprachiasmatic nucleus or nuclei, also known as the master pacemaker in mammalian species. A bilateral set of nuclei positioned

in the anterior ventral hypothalamus. Essential for entraining extra-SCN central and peripheral oscillators to the prevailing light-dark

cycle via photic input from the retina.

Shift work Work scheduled so that it occurs outside of the traditional work schedule of 9:00 a.m. to 5:00 p.m., or 6:00 a.m. to 6:00 p.m., depending on

definition. Various forms of shift work exist, such as early morning, evening, or night shifts, as well as rotating shifts.

Ultradian rhythm A recurrent cycle or period with a period significantly shorter than 24 h—e.g., a 2-h rhythm would exhibit 12 cycles within a circadian

(24-h) rhythm.

limb of the clock is composed of the basic helix-loop-helix transcription factors (TFs) CLOCK (or its paralogue, NPAS2) and BMAL1.

These drive expression of their own repressors (PER and CRY) in the

negative limb in a cycle that repeats itself every 24 h (Fig. 485-1). A

second short feedback loop involves CLOCK/BMAL1-mediated transcription of the retinoic acid–related orphan nuclear receptor families

ROR and REV-ERB, which activate and repress Bmal1 transcription,

respectively. Rhythmic posttranslational regulation of the stability

and degradation of core clock TFs occurs via events such as phosphorylation by casein kinase 1 epsilon (CK1ε) and casein kinase 1 delta

(CK1δ) and ubiquitination by FBXL3 and FBXL21. In addition to the

circa 24-h oscillation of core clock genes, a wide array of downstream

clock-controlled genes (CCGs) exhibit broad rhythmic amplitude in

expression, ultimately giving rise to rhythmic physiologic processes.

The importance of localized clock gene expression has been demonstrated by genetic animal studies, such as with targeted ablation of

Bmal1, the only clock gene that lacks a known functional paralog.

Deletion of Bmal1 either in the whole brain or in regions that span

the brain region that coordinates circadian rhythms—the SCN—cause

behavioral arrhythmicity, even when genetic ablation occurs in adult

life. Conversely, restoring Bmal1 expression specifically in brain in

adult CLOCK mutant mice rescues behavioral locomotor rhythms.

Of note, whereas the protein CLOCK normally heterodimerizes with

BMAL1, NPAS2 is able to functionally substitute for CLOCK within

the pacemaker neurons; thus, while mice lacking either Clock or Npas2

genes maintain rhythmicity, mutants lacking both CLOCK and NPAS2

lack circadian rhythms in locomotor activity.

A major transformation in our understanding of circadian biology

came with the discovery that the molecular clock network is present

not only in the SCN but also within most peripheral tissues, as well as

in extra-SCN neurons in the brain. In primates, ~82% of all proteincoding transcripts exhibit daily (sleep-wake) rhythms in some tissue

or other. In rodents studied under constant conditions, ~3–16% of

the transcriptome in a given tissue displays 24-h rhythms in mRNA

expression levels, even though the repertoire of such genes varies substantially between tissues, in accordance with tissue-specific functions.

The core clock feedback loop and the induction of transcriptional CCG

rhythms also involves epigenetic mechanisms such as conformational

chromatin dynamics, histone acetylation, and DNA methylation.

Conversely, posttranscriptional events such as RNA polyadenylation,

nuclear shuttling, and mRNA translation also exhibit circadian variation, further increasing the repertoire of rhythmic regulation at a

cellular level.

■ ANATOMIC ORGANIZATION OF THE CIRCADIAN

CLOCK NETWORK

The molecular circadian feedback loop is synchronized with sunrise

each day by photosensitive melanopsin-expressing neurons within

3802 PART 20 Frontiers

the retina. These neurons provide input to the SCN via the retinohypothalamic tract (RHT), allowing mammals to maintain coherent

organismal rhythms in line with the external light/dark cycle. Of note,

mutations in several of these clock genes are associated with impaired

circadian rhythms and physiology in humans (see “Primary Pathologies of the Circadian System” below).

Understanding the circuit organization of the circadian clock within

the brain is increasingly relevant in understanding how the master circadian pacemaker center within the SCN regulates feeding, sleep-wake

activity, endocrine processes, and energy expenditure and metabolism

(Fig. 485-2). Identification of the SCN as the master pacemaker was

first established by the observation that SCN lesioning induced complete loss of rhythms of locomotor activity, drinking behavior, and

endocrine hormone secretion. The ventral “core” region of the SCN,

which is composed of neurons producing vasoactive intestinal polypeptide (VIP), receives photic information directly from the retina

through the RHT. At the molecular level, circadian gene transcription

is induced within the SCN through the initial activation of immediate

early genes, such as Per1, Per2, c-fos, and jun. Cells within the “core”

region of the SCN then signal primarily via γ-aminobutyric acid

(GABA)-ergic neurotransmitter release to synchronize the cells within

the “shell” region of the SCN, which produce arginine vasopressin

(AVP), the most important neuropeptide for maintaining intra-SCN

synchronicity.

The SCN communicates to extra-SCN and peripheral clocks

through both secreted factors and neuronal projections. The former

was elegantly proven by the ability of SCN grafts to partially restore

locomotor rhythms in SCN-lesioned animals. Efferent nerve outputs

arise both from the AVP-producing shell region of the SCN and the

VIP-predominated core. The SCN projects to several hypothalamic

and thalamic regions, including the median preoptic nucleus (MPO),

the subparaventricular zone (SPZ), the dorsomedial hypothalamus

(DMH), the paraventricular nucleus of the hypothalamus (PVH), and

the paraventricular nucleus of the thalamus (PVT). Some of these

regions, in turn, regulate output to both sleep- and wake-promoting

regions, as well as to regions involved in regulation of autonomic, body

temperature, and hormonal rhythms, as well as feeding. The SCN is

thereby thought to promote sleep in part through the transmission

of neural signals that terminate in the sleep-promoting ventrolateral

preoptic nucleus (VLPO), i.e., one of the brain regions that is active

during sleep. In contrast, the SCN promotes wakefulness during the

active phase by transmission of neural signals that—by passing through

regions such as the SPZ and the DMH—terminate in wake-promoting

regions, including the locus coeruleus, lateral hypothalamic nucleus,

ventral tegmental area, and dorsal raphe nucleus.

The SCN also signals via noradrenergic fibers to the pineal gland,

thereby regulating the circadian production of the hormone melatonin. SCN control of the nighttime rise in pineal melatonin release (in

both diurnal and nocturnal animals) is mediated through a pathway

involving the PVH. Of note, artificial light at night delays the secretion

of melatonin, ultimately affecting sleep (see “Endocrine Systems Regulated by the Circadian Clock” below). Melatonin plays a complex role

in the circadian system since the MT1 and MT2 melatonin receptors

are expressed in the SCN itself; thus, melatonin feeds back to modulate

circadian outputs to other cells in the brain and body.

Neuronal output from the SCN also reaches the periphery, i.e., to

the adrenal glands, the liver, and the pancreas. The SCN produces

rhythmic variation in multiple neuroendocrine axes, producing daily

rhythms of gonadotropin, thyrotropin, and somatotropin. Prominent HPA axis rhythms ultimately give rise to daily variation in

diverse pathways essential for hemodynamic stability, metabolism, and

inflammation. These rhythms originate with SCN control of corticotropin-releasing hormone (CRH)–producing cells in the PVH, which

may regulate sleep as well as induce daily oscillations of both pituitary

adrenocorticotropic hormone (ACTH) and adrenal cortisol. Highlighting the importance of SCN output for peripheral rhythms, there is a

dramatic reduction in the number of transcripts that exhibit circadian

rhythms in the liver following SCN ablation in mice. Nonetheless,

when the autonomous clock in the liver is ablated in mice, some key

clock transcripts such as Per2 are still able to cycle as long as the core

body temperature rhythm persists. Whereas the SCN is exclusively

entrained by light, meal timing is able to signal circadian time directly

to peripheral tissues such as the liver. Thus, shifted meal timing as

occurs during shift work or jetlag can uncouple peripheral clocks from

the central pacemaker. In comparison to peripheral tissue clocks, the

SCN is also resistant to phase shifts induced by temperature. This is

consistent with the concept that the SCN generates the core body temperature rhythm as one of the major mechanisms to signal circadian

time to peripheral clocks.

■ ENTRAINMENT AND MEASUREMENT OF THE

CIRCADIAN SYSTEM

Under normal light-dark cycles, the circadian system is corrected or

“entrained” on a daily basis, producing diurnal rhythms of 24 h. Such

signals of entrainment are called zeitgebers (German for “time-giver”

signals) and include light exposure, meal timing, and activity patterns.

Light serves as the dominant zeitgeber for the circadian system, and a

breakthrough in understanding photoentrainment in mammals came

with the discovery of the melanopsin system, which is composed of a

specialized class of photosensitive retinal ganglion cells that expresses

CRYs

P FBXL3

CRYs

FBXL21

CK1ε/δ

PERs

P

CRYs

P

E-box

BMAL1 CLOCK

Rev-erbα/β

RORα/γ

RRE Bmal1

Degradation

Clock-controlled genes

Stabilization

FIGURE 485-1 Central clock molecular mechanism. The core molecular clock machinery in mammals is encoded by interlocking transcription-translation feedback loops

that oscillate with ~24-h periodicity. The transcription factors CLOCK and BMAL1 heterodimerize to drive transcription of downstream clock-controlled target genes

containing E-box enhancer elements. Among these, the PER and CRY proteins multimerize and inhibit CLOCK/BMAL1, while RORs and REV-ERBs activate and inhibit,

respectively, Bmal1 transcription, resulting in rhythmic oscillations of clock-controlled and downstream target genes.

3803The Role of Circadian Biology in Health and Disease CHAPTER 485

the blue light–sensitive photopigment melanopsin in the inner retina,

separate from the photoreceptive rods and cones. Blue light around this

wavelength (~480 nm) suppresses melatonin, i.e., melatonin levels are

normally low during the day, and promotes subjective and objective

(electroencephalography-assessed) wakefulness.

The ability of light to entrain the circadian system functions according to a so-called phase response curve (PRC). When light exposure

occurs prior to the critical phase of the core body temperature (CBT),

defined by the CBT’s minimum, light produces a phase delay in the

circadian rhythm. Conversely, light exposure after this critical period

causes phase advances. The circadian system can respond even to

small changes in light intensity (e.g., dim light at ~100 lux can produce

half of the phase delay compared with an almost 100-fold greater light

exposure). This responsiveness has been found to be highly individual,

varying several tenfold. This is in part due to genetic variation, as

variants in clock genes can modulate the responsiveness of the human

circadian system to light.

When an organism is placed in an environment without zeitgebers,

the circadian rhythm is said to free-run, as it relies on the endogenous

rhythm of the circadian system. In humans, the study of endogenous

circadian rhythms can be achieved by using a so-called constant routine that eliminates the risk of masking by factors such as sleep. In

these paradigms, subjects are kept awake in a constant semirecumbent

posture, meals are provided on an hourly basis, light is constantly

kept below the level that phase shifts the SCN, and circadian rhythms

are assessed by frequently measuring CBT, melatonin, or peptidergic hormone rhythms over the course of 24 h or longer. In animals,

circadian rhythms are instead studied by examining behavior and

physiologic responses after 30–36 h of complete darkness, and endogenous rhythms are assessed by measuring voluntary locomotor activity.

From these measurements, key properties of the circadian system

can be ascertained, such as period length (peak-to-peak or troughto-trough time), amplitude (peak-to-trough difference), and phase

(timing of peak or trough in relation to a reference point) (Fig. 485-2).

These studies have revealed that the endogenous human circadian

clock runs with a period length of approximately 24.2 h (compared

with that of mice, which runs at ~23.5 h, depending on the strain).

Evidence indicates that human females may have a slightly shorter

circadian clock (24.1 vs 24.2 h), and many circadian parameters have

been found to exhibit differences that are dependent on biological sex.

Notably, interindividual variability in the endogenous circadian period

length is further diversified by the existence of genetic polymorphisms

in clock genes (see below). These gene variants can confer extremes

in the endogenous circadian period as well as phase; the latter can be

advanced or delayed by ~3–4 h in each direction. This is due both to

altered circadian rhythms at the cell level and to altered SCN responsiveness to entrainment by light. For instance, PER3 exists in a variablenumber, tandem-repeat polymorphism. Individuals homozygous for

a PER3 5/5 genotype have been reported to be more responsive than

PER3 4/4 homozygous individuals to the melatonin-suppressing effect

of evening blue light exposure.

Using specifically-developed questionnaires to establish preferred

sleep-wake timing, individuals can be categorized into so-called

morningness-eveningness types or chronotypes. The most commonly

used questionnaires are the Horne-Östberg Morningness-Eveningness

Questionnaire (MEQ) and the Munich ChronoType Questionnaire

∆ Amplitude

(Night eating, insulin resistance)

∆ Period

(high fat)

∆ Phase

(Shift work)

Original phase New phase

Environmental

light cycle

Internal

circadian time

Environmental inputs and

internal circadian organization

Behavioral and

physiologic outputs

Circadian

disruptors

Environmental

nutrient cycle

fasting/feeding

Brain Clocks

Extra-SCN

LHA

ARC PIT

PVN

SCN

SCN

Non-autonomous

circadian control

Peripheral Clocks

Vasculature

Intestine

Pancreas

Adrenals

Fibroblasts

Hematopoetic

Liver

Muscle

Adipose

Autonomous

circadian control

period

amplitude

phase

Glucose homeostasis

Lipogenesis

Oxidative metabolism

Mitochondrial respiration

Xenobiotic detoxifixation

Cytokine production

Vascular tone

Hemostasis

Stress response

Thermogenesis

Incretin production

DNA damage/repair

Environmental

light/dark

cycle

Sleep/wake

Feeding/fasting

Energy expenditure

Glucose homeostasis

FIGURE 485-2 Central and peripheral clocks coordinate environmental cues with behavior and physiologic outputs. Light entrains the master pacemaker neurons in

the suprachiasmatic nucleus (SCN), which subsequently synchronizes extra-SCN and peripheral clocks. Brain clock output includes sleep-wake, fasting-feeding, and

energy expenditure cycles, while peripheral clock output includes a wide range of physiologic processes, including glucose homeostasis, oxidative metabolism, cytokine

production, and stress response. The right column indicates different ways that circadian disruptors, such as diet, shift work, or other circadian rhythm sleep disorders, may

impact the clock—i.e., by changing circadian period, phase, or amplitude.

3804 PART 20 Frontiers

(MCTQ). A composite MEQ score allows grouping into five categories

that range from definite morning-type to evening-type individuals, for

instance, based on preferred waking time. In contrast, the MCTQ centers on the midpoint of sleep as a circadian marker, queries age and sex

across a range of geographical locations, and can be used to ascertain

differences between socially imposed sleep patterns (e.g., on working

days) and sleep patterns on free days (the difference constituting socalled social jetlag). According to MCTQs obtained from primarily

European populations, ~1% of the general population goes to bed

before 10:00 p.m. and ~8% after 3:00 a.m. Differences in chronotype are

linked to altered circadian timing, including peak levels of melatonin,

which can vary by up to 4 h between extreme morning and evening

types. Extreme chronotypes have also been shown to be linked to

various traits; i.e., low morningness scores have been associated with

greater tolerance to shift work.

Melatonin is one of the most commonly used peripheral markers of

an individual’s circadian rhythm, reflecting the rhythmic function of

the SCN. Circadian rhythms of melatonin can be measured in saliva

or plasma, whereas 6-sulphatoxymelatonin (aMT6S), a metabolite

generated from the breakdown of melatonin, can also be measured in

urine. Accurate estimations of melatonin rhythms are often obtained

by analyzing the dim light melatonin onset (DLMO), which as the

name implies does not require an entire 24-h sampling, making this

marker useful in both the clinical and research settings. In normally

entrained individuals, the DLMO can be used to ascertain whether an

individual’s circadian rhythm is phase advanced or delayed, and this

onset typically occurs ~2 h before the onset of sleep. The midpoint of

sleep—the main marker used by the MCTQ—also strongly correlates

with melatonin onset.

The CBT is also often utilized as an indicator of the circadian

rhythm. Even though the outcome is more variable when using the

CBT, it usually correlates well with the phase obtained using the melatonin rhythm. The CBT, however, can be masked by factors such as

sleep, food intake, and activity. CBT can be recorded and registered

wirelessly with relative ease. In humans, CBT can be recorded via rectal

thermometers or probes that are swallowed to pass through the gastrointestinal tract. When humans are studied under normal conditions

with normal lighting and sleep duration from 2300 to 0700 h, the CBT

reaches around 37.2°C by 0900 h, and from there, it continues to rise

slowly until it reaches 37.4°C around 11 h later. The CBT then drops to

the daily low of 36.5°C in the early morning (0400 h).

Given the interrelationship between the circadian system and sleepwake systems, researchers have developed paradigms that uncouple

the circadian system from sleep-wake states, enabling the study of the

contribution of the circadian system to investigated parameters across

the entire sleep-wake cycle. These paradigms are known as “forced

desynchrony” protocols and involve enforcing a significantly shortened

(e.g., 20 h) or prolonged (e.g., 28 h) day length upon individuals. These

protocols thus attempt to approximate what occurs during rotating

shift work or “jetlag,” e.g., when travel across several time zones suddenly shifts the light-dark and behavioral cycles drastically away from

the entrained 24-h rhythm. As described below, forced desynchrony

protocols have contributed to uncovering how the circadian system

regulates parameters such as cognitive performance, subjective alertness, and metabolic and cardiovascular health.

■ PRIMARY PATHOLOGIES OF THE CIRCADIAN

SYSTEM

An overarching term for disorders of the circadian system is circadian

rhythm sleep disorders (CRSDs). Several of these disorders are quite

common and have become increasingly recognized as important factors in a number of conditions. A unifying feature of CRSDs involves

a mismatch between subjective behavioral and physiologic rhythms

with the environmental light-dark or social activity-rest cycles (i.e., the

body clock is out of sync with the external light-dark cycle). CRSDs

can arise either due to misalignment of an exogenous environmental

factor, such as light, with the intrinsic circadian cycle or due to misalignment of the activity-rest cycle in relation to endogenous circadian

timing (e.g., shift work or jetlag). In addition to such environmental

or exogenous conditions causing circadian disruption, in some cases,

intrinsic circadian timing is altered in relation to the external environment, as in the case of endogenous circadian disorders such as those

caused by mutations in core clock genes. Under conditions of intrinsic

circadian abnormalities, it is often exceedingly difficult for individuals

suffering from CRSDs to try to properly realign themselves, and these

disorders often result in adverse effects such as excessive sleepiness or

depressed mood. Societal and economic consequences are also common; these can result in the individual being unable to maintain a job

or attend school at regular hours. The criteria for CRSDs based on the

International Classification of Sleep Disorders (ICSD) are shown in

Table 485-2.

Animal models have greatly advanced our understanding of how

core molecular clock components contribute to maintaining normal

sleep-wake/rest-activity cycles (Table 485-3). For example, Clock∆19/∆19

mice have reduced total sleep duration and less induction of rapid

eye movement (REM) sleep in response to sleep deprivation. Further,

mice that lack Bmal1 have increased total sleep time, but it is more

fragmented and lacks clear 24-h sleep-wake rhythms, and mice lacking

the repressors Cry1 and Cry2 are not only arrhythmic but spend more

time in non-REM sleep. Finally, while ablation of the circadian gene

Dbp does not alter the specific duration of sleep stages, it does lead to

an altered circadian sleep-wake distribution, with more sleep during

the normal wake period and vice versa. Consistent with a key role of

clock genes in regulating sleep-wake behavior, human genetic studies

of twins have found that up to half of the variation in diurnal preference is heritable. Established genetic variants associated with diurnal

preference and circadian sleep disorders are listed in Table 485-4.

Delayed Sleep Phase Disorder Delayed sleep phase disorder

(DSPD; or delayed sleep-wake phase disorder [DSWPD]) is one of

the more common circadian rhythm sleep disorders. The true prevalence is not fully known but may range from 0.2–16% depending

on definition used, and the condition is more common in younger

age groups. DSPD is characterized by chronic and significant delays

in both sleep onset and wake times compared to normal “socially

acceptable” sleep-wake hours (i.e., scoring as “extreme night owls” on

morningness-eveningness preference tests). Rhythms of parameters,

such as CBT and melatonin levels in plasma and urine, are likewise

often delayed, and the circadian period (tau) may be longer in DSPD.

Onset of DSPD most commonly occurs during adolescence or early

adulthood. While the precise etiology of DSPD is not well established,

it has been associated with polymorphisms within the circadian clock

genes CLOCK, PER3, and CRY1. As an example, the latter mutation

has been observed at a frequency of ~0.6%. An integrated behavioral

and pharmaceutical therapeutic approach has been found to be most

effective at treating individuals with DSPD. Such treatments include a

combination of bright-light therapy soon after waking in the morning

(and/or dark-room therapy in the evening) and melatonin administration in the evening several hours prior to the onset of sleep. These

approaches aim to realign endogenous circadian rhythms with the

desired sleep-wake schedule. As individuals suffering from DSPD also

phase delay more rapidly, this explains why attempts to phase advance

their sleep schedule can be difficult, as well as why relapse can easily

occur after initial treatment.

TABLE 485-2 Criteria for Circadian Rhythm Sleep Disorders

CRITERIA DESCRIPTION

A A persistent or recurrent pattern of sleep disturbance due

primarily to one of the following:

• Alterations of the internal circadian timekeeping system.

• Misalignment between endogenous circadian rhythms and

exogenous factors that affect the timing or duration of sleep.

B A circadian-related sleep disruption that leads to insomnia,

excessive daytime sleepiness, or both.

C A sleep disturbance that is associated with impairment of social,

occupational, or other areas of functioning.

3805The Role of Circadian Biology in Health and Disease CHAPTER 485

Advanced Sleep Phase Disorder Another circadian rhythm

sleep disorder whereby one gets the correct amount and quality of

sleep but at a shifted time is advanced sleep phase disorder (ASPD;

or advanced sleep-wake phase disorder [ASWPD]). The prevalence of

this disorder may be <1%, but the condition may be underreported,

given that it may cause fewer conflicts with societal demands (i.e.,

9-to-5 schedules) compared with conditions such as DSPD. Individuals with ASPD experience an advance in their major sleep episode in

relation to the desired sleep-wake times. Thus, this disorder typically

results both in very early evening bedtimes and morning awakenings

(e.g., “extreme early birds”), resulting in reduced quality of life due to

excessive sleepiness during the early evening, even in social situations.

Individuals with ASPD also have phase-advanced temperature and

melatonin rhythms in parallel with their earlier sleep onsets. ASPD

occurs more often in older individuals, although early-onset autosomal

dominant familial variants (familial advanced sleep phase syndrome

[FASPS]) have also been associated with mutations in either the PER2

or the casein kinase 1δ (CK1δ) gene. PER2 is critical for SCN resetting

by light, and the identification of PER2 mutations in familial ASPD was

the first instance in which clock genes were tied to a CRSD. Such mutations have been found to shorten the endogenous circadian period to

~23.3 h compared with the normal 24.2-h period length. Accordingly,

ASPD can be distinguished from other noncircadian sleep disorders

by an early onset of dim-light melatonin secretion. Similar to DSPD,

polysomnography and actigraphy are not required for diagnosing

ASPD, although actigraphy, preferably for 14 days or longer, may be

significantly more feasible for long-term analysis of circadian timing

of sleep. Treatments for ASPD include bright light or blue-enriched

TABLE 485-3 Animal Models of Genetic Circadian Disruption

AVERAGE CIRCADIAN TIME OF PEAK TRANSCRIPT LEVEL

GENE SCN PERIPHERY ALLELE MUTANT PHENOTYPE

Bmal1 (Arntl) 15–21 22–2 Bmal1−/− Arrhythmic

CK1δ (Csnk1d) No rhythm No rhythm Csnk1d+/– 0 to 0.5-h shorter period

CK1ε (Csnk1e) No rhythm No rhythm CK1etau 4-h shorter period

CK1ε — — CK1e−/− 0.2- to 0.4-h longer period

Clock No rhythm 21–3 Clock−/− 0.5-h shorter period

— — — ClockΔ19/Δ19 4-h longer period/arrhythmic

Clock/Npas2 — — Clock−/−/NPAS2−/− Arrhythmic

Cry1 8–14 14–18 Cry1−/− 1-h shorter period

Cry2 8–14 8–12 Cry2−/− 1-h longer period

— — — Cry2A260T 0.2-h shorter period

Dbp — — Dbp−/− 0.5-h shorter period

Npas2 N/A 0–4 Npas2−/− 0.2-h shorter period

Per1 4–8 10–16 Per1−/− 0.7-h shorter period

— — — Per1brdm1 1-h shorter period

— — — Per1ldc 0.5-h shorter period/arrhythmic

Per2 6–12 14–18 Per2brdm1 1.5-h shorter period/arrhythmic

— — — Per2ldc Arrhythmic

Per3 4–9 10–14 Per3−/− 0 to 0.5-h shorter period

Rev-erbα (Nr1d1) 2–6 4–10 Rev-erba−/− 0.5-h shorter period/disrupted photic

entrainment

Rorα 6–10 Arrhythmic/various staggerer 0.5-h shorter period/disrupted photic

entrainment

Rorb 4–8 18–22 Rorb−/− 0.5-h longer period

Rorg N/A 16–20/various Rorg−/− Normal behavior

Note: Normal circadian rhythms of circadian clock and related genes, with description of circadian phenotype in mutant mice.

Abbreviation: N/A, not applicable.

Source: Adapted from Hum Mol Genet 15:R271, 2006, and Adv Genet 74:175, 2011.

TABLE 485-4 Mutations and Gene Variants Linked to Sleep-Wake Disorders and Diurnal Preference

GENE POSITION POPULATION SYNDROME/SLEEP PREFERENCE

hCKIe S408N Japanese Protection against DSPS

hCKIg T44A Pedigree FASPS

hCKIΔ H46R Pedigree FASPS

hCLOCK T3111C (3′-UTR) European Eveningness

hCRY2 A260T Pedigree FASPS

hPER2 S662G (missense mutations in CKIε binding region) Pedigree FASPS

hPER2 C111G (5′-UTR) British Extreme morningness

hPER3 P415A/H417R Pedigree FASPS and seasonal affective disorder

hPER3 G647 Swedish/Finish/Austrian/German Morningness

hPER3 G647, P864, 4-repeat, T1037, R1158 Japanese DSPS

hPER3 Increased repeats (exon 18, 54 bp) Brazilian DSPS

hVIP rs9479402 (gene variant 54 kb upstream of VIP) European (>97% European ancestry) Morningness

Abbreviations: DSPS, delayed sleep phase syndrome; FASPS, familial advanced sleep phase syndrome.

3806 PART 20 Frontiers

phototherapy in the evening hours to delay the phase of the circadian

clock to a later hour.

Shift Work Sleep Disorder Given the increased prevalence of

shift work in today’s 24/7 society and the accumulating evidence for

increased incidence of sleep and metabolic disorders, including obesity,

type 2 diabetes, cardiovascular disease, and cancer, in shift workers,

the need to develop effective treatments for shift work sleep disorder

(SWSD) is increasingly important. SWSD is at its core defined by the

primary symptom of either insomnia or excessive sleepiness, arising

as a result of work that usually is scheduled during the habitual hours

of sleep or comprises irregular work hours. The symptoms may result

from recovery of sleep having to consume a large proportion of the

individual’s free time, which may produce negative social consequences

such as difficulties maintaining social relationships. Older individuals

are typically at an increased risk of SWSD due to age-associated decline

in the ability to maintain sleep during the time of day that would normally constitute the wake period. Since the symptoms likely arise from

a misalignment of sleep-wake rhythms with the external light-dark

cycle, therapeutic approaches aim to realign endogenous circadian

rhythms with the sleep cycles dictated by work. In addition to optimizing the sleep environment at home to minimize disruptions, timed

bright light therapy can help individuals with SWSD—i.e., for night

workers, intermittent bright light exposure during the night and avoidance of bright light during the morning, even on days off, has been

shown to improve sleep and feelings of alertness. Melatonin prior to

bedtime may also help improve symptoms of SWSD. Genetic screening

combined with chronotype questionnaires may become useful tools

for determining whether a given individual is suited for shiftwork. For

instance, a twin study indicated that a genetic variant of the circadian

gene DEC2 was associated with reduced sleep duration and shorter

recovery sleep following extended sleep deprivation. More studies may

reveal additional genetic variants that confer an advantage to repeated

phase advances and phase delays as typically occurs in shift work.

Irregular Sleep-Wake Rhythm Damage to the SCN can produce

arrhythmicity in animals and is thought to be one of the possible

underlying reasons for the temporally disorganized sleep-wake pattern

that characterizes the disorder known as irregular sleep-wake rhythm

(ISWR). Other contributing factors may be a reduced responsiveness

to entraining signals such as light and physical activity, as well as a

decreased exposure to such signals as often occurs with increasing age

due to, for example, increased risk of poor health and impaired mobility. Whereas the total sleep time per 24 h may be comparable, there is

a relative absence of a circadian pattern to the sleep-wake cycle. Sleep

timing throughout the sleep-wake cycle can be shortened—sometimes

close to randomly distributed—instead of occurring in several distinct

bouts. ISWR is often associated with neurologic impairment, foremost

Alzheimer’s disease in older age; however, ISWR can also occur in

individuals with poor sleep hygiene. The most effective treatments for

ISWR involve not pharmacotherapy, but rather multimodal interventions such as increased light exposure, improved sleep hygiene, and

promotion of social and physical activities.

Non-24-h Sleep-Wake Rhythm Disorder Individuals with

non-24-h sleep-wake rhythm disorder (“non-24”), otherwise known

as free-running disorder (FRD), have endogenous circadian rhythms

that are not synchronized with the external 24-h day-night cycle due

to an inability to readjust the circadian clock to the 24-h day on a daily

basis. This most commonly occurs in individuals who are completely

blind (i.e., lacking all photoreceptors) since they are unable to respond

to light cues that normally would reset the endogenous circadian clock

on a daily basis (although the condition has also been reported in

sighted individuals). Instead, the sleep-wake period length corresponds

to the individual’s endogenous circadian rhythms, which are typically

slightly longer than 24 h, thereby shifting sleep and wake cycles over

time in relation to the light-dark cycle. Instead of sleeping at the same

time each day, their sleep time would gradually be delayed each day

until their sleep period literally goes “around the clock.” Depending on

the individual’s endogenous rhythm, the individual will take a given

number of days to realign his or her endogenous phase (in a 360°

phase plot) with the zero time point in the exogenous 24-h light-dark

cycle. For example, if an individual has an endogenous 24.5-h rhythm,

it would take that individual 48 days to cycle from one cycle to the

next. Because of this chronic cycling, prominent symptoms of non-24

include sleep-wake cycle disruption (insomnia and daytime sleepiness), impaired alertness and mood levels, and severe difficulties partaking in normally scheduled work, school, or social activities. Non-24

can be diagnosed following diurnal analysis of an individual’s melatonin or cortisol rhythms, in combination with analyses of sleep diaries

where the sleep onset and offset can be visualized over time to identify

the free-running period. Treatments for sighted non-24-h patients

include a combination of bright light therapy with appropriately timed

melatonin administration, whereas melatonin and dual melatonin

(MT1 and MT2) receptor agonist administration in completely blind

non-24-h patients has been shown to entrain free-running rhythms

and improve symptoms.

Jetlag Most have experienced symptoms associated with jetlag,

including insomnia, daytime sleepiness, and fatigue, when traveling

from one time zone to another, as one’s endogenous circadian rhythms

are not yet aligned, or entrained, to the new external light-dark cycle.

This is due to the slowness of the circadian system to adapt to the new

time zone: typically, the human circadian system is able to shift up to

~1.5 h a day in the westward direction (i.e., a phase delay), whereas

it shifts more slowly (up to ~1 h daily) with eastward direction of

travel (i.e., achieving a phase advance). Importantly, the symptoms are

distinguished from the more short-lived symptoms that can partially

result from exposure to traditional airplane cabin conditions, including

abdominal distention, dependent edema, muscle cramps, headaches,

nausea, and intermittent dizziness. Usually, symptoms of jetlag abate

within the first couple of days after traveling and may present themselves after a first night of good sleep (which is more dependent on

a high buildup of homeostatic sleep pressure). Older individuals (age

>50) appear to be more at risk. While symptoms are transient, therapeutic approaches can alleviate or temper some of the side effects of

travel by hastening synchronization of the internal and external circadian cycles. Behavioral treatments include appropriately timed brightlight exposure and avoidance of bright light during the nighttime in

the new destination, while pharmacologic approaches include timed

melatonin administration before bedtime both prior to and following

travel, resulting in improved sleep quality and decreased night waking.

Social Jetlag Individuals with a late chronotype are prone to suffer

from “social jetlag,” a phenomenon in which individuals are forced to

awaken at a point at which their bodies are entrained to be asleep due

to discrepancy between alignment of social and biological time. Social

jetlag can be estimated using questionnaires, such as the MCTQ, to

compare sleep timing on nonfree compared with free days. This has

established that a large proportion of the European population suffers

from 2 or more hours of social jetlag. Chronic social jetlag is associated with an increased risk of developing obesity and the metabolic

syndrome, as well as with greater alcohol consumption, smoking, and

poorer academic performance in students.

The aforementioned categories of defined clinical circadian disorders have been traditionally established based on consideration of the

endogenous behavioral and physiologic cycles (primarily of melatonin

and temperature) with the external 24-h light-dark cycle. In the following sections, we build on the concepts of circadian behavioral disorders to consider new and emerging insight into the role of circadian

disruption in organismal homeostasis (Figs. 485-3 and 485-4) and the

availability of genetic strategies to dissect the interrelationship between

clock function, health, and disease.

■ ROLE OF THE CLOCK SYSTEM IN PHYSIOLOGY

Endocrine Systems Regulated by the Circadian Clock In

addition to regulation of behavioral rhythms such as sleep-wake and

fasting-feeding cycles, the circadian clock also regulates rhythms

of the endocrine system. Cortisol rhythms are regulated through a