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Leading Edge
Essay
Modeling Human Nutrition
Using Human Embryonic Stem Cells
1, 1
DannyBen-Zvi * and Douglas A. Melton
1
DepartmentofStemCellandRegenerativeBiology,HarvardStemCellInstitute,HarvardUniversity,7DivinityAvenue,CambridgeMA,USA
*Correspondence: benzvi@fas.harvard.edu
http://dx.doi.org/10.1016/j.cell.2015.02.039
Nutrition presents unanswered scientific questions of high public health importance. We envision
model systems composed of interacting gastrointestinal and metabolic tissues derived from
human embryonic stem cells, populated by gut microbiota. The culture will be embedded in 3D
scaffolds, creating a controlled experimental system that enables tissue sampling and imaging.
Introduction autoimmune diseases? Is it the high car- respond to distinct nutritional challenges,
In the 19th century, important advances in bohydrate, high fat content, or both, of their responses to food differ; indeed, our
physiologyarosefromanunusualrelation- western diets that leads to obesity and diet and lifestyle are strikingly different
shipbetweenthephysicianWilliamBeau- metabolic syndrome? (Antico et al., from those of model organisms. More-
mont and his patient Alexis St. Martin. 2012; Nature Editors, 2014; Feinman over, most model organisms have limited
St. Martin received a shotgun wound that et al., 2014; Key et al., 2006)? Even in genetic and environmental variability. On
left him with a sizeable opening in his cases with conclusive epidemiologic the other hand, primary and immortalized
stomach that did not close. Beaumont data, the underlying mechanisms that human cell lines are easy to manipulate,
availedhimselfofSt.Martin’suniquecon- link nutrition and pathology are not are amenable to imaging and screening,
dition and conducted some of the first entirely clear: how does obesity lead to and provide highly reproducible results.
digestion experiments. For 10 years he development of insulin resistance? How Yet,immortalizedcelllinesdiffermetabol-
continuedthis research combining in vivo does a Mediterranean diet prevent car- ically from cells in vivo and are limited in
and in vitro approaches. For example, by diovascular disease (Estruch et al., 2013; their ability to teach us about the function
tying food to a string and inserting it Qatanani and Lazar, 2007; Ye et al., of organs and the intra and inter-organ
through the hole in St. Martin’s stomach 2012)? These unanswered questions signaling.
and retrieving it later, Beaumont was lend themselves to nutritional myths and Digestion is a complex process per-
able to retrieve the food and observe inconclusive recommendations that have formed physically and chemically by the
digestion in real time. He also removed becomeall too commonplace in the daily gastrointestinal (GI) organs and exocrine
gastric juice from St. Martin’s stomach news. As a result, the public is easily glands together with gut bacteria. Food
watched it digest food in a test tube, confused by opposing views supported is sensed in the gut by enteroendocrine
providingnewinformationonthemechan- by scientific evidence (Malik and Hu, cells that prepare the body for the
ical and chemical nature of digestion. 2007; Matarese and Pories, 2014; Willett incoming meal: For example, the hor-
Obviously,thescientificcommunityhas and Stampfer, 2013). mone Cck regulates the release of
come a long way since then, with new Nutritional questions may be easy to pancreatic enzymes, Pyy signals to neu-
methodstostudyphysiologyandnutrition pose, but they are difficult to answer for rons that regulate satiety and behavior,
using cell lines, model organisms, and reasons we outline below. Here, we pro- Glp1 sensitizes insulin-secreting cells
endoscopic views of the human digestive pose a stem-cell-centric point of view in anticipation for the increase in blood
system. Nutritional scientists studying andsuggestthathumanstemcellmodels glucose (Psichas et al., 2015). The im-
microbiology, physiology, neurobiology, of digestive processes could be a power- mune system sustains a symbiotic rela-
immunology, epidemiology, genetics, ful experimental system to study the tionship with gut bacteria necessary for
and behavior have made substantial physiology of digestion and nutrition in digestion (Round and Mazmanian, 2009),
progress in understanding the biology of the future. and the enteric and nervous systems
food. regulate gut motility, behavior, and blood
Nonetheless, we have yet to compre- Challenges in Nutritional Science circulation through chemical and physical
hensively answer some major nutritional Many discoveries in physiology have sensing of food. Metabolites themselves
questions of wide public interest. For been made possible by the use of model act as signaling molecules, capable of
instance, is being vegetarian beneficial organisms, including fish, rodents, and binding nuclear receptors such as the
to one’s health, and if so, how? Is organi- flies. Advanced experimental tools were PPARproteins. All cells consume metab-
cally farmed food healthier or only more developed to study these organisms, olites and change their cell biology in
expensive? How does the introduction of and they will continue to provide invalu- accordance with food availability, linking
new crops and cooking methods affect able data for many years to come. How- nutrition to every process in the body
our health? Can nutrition reduce risk for ever, since each species evolved to and making nutrition a Gordian knot.
12 Cell 161, March 26, 2015 ª2015 Elsevier Inc.
Variability between individuals must a new range of research avenues on equivalent of blood: a medium that will
be taken into account. Not only do we understanding human gut biology and support the culture of many cell types
have different eating habits and taste disease through the use of hESCs and and transport biomolecules between
preferences, but we also vary in our iPSCs. different organs. This bottleneck must be
ability to digest and metabolize food. Other cell types can also be differ- relieved before a modular, multi-organ
Metabolic variability of individuals is entiated from hESCs. Insulin-secreting system can be incorporated to study
largely unexplored and may be affected b-cells can be grown from hESCs in clus- digestion and nutrition, or any other
by numerous factors, including culture, terscomparableinsizetoanisletofLang- multi-organ process. Nonetheless, re-
psychology, genetics, gut microbiome erhans (Pagliuca et al., 2014). Similarly, a searchers have been able to generate
composition, epigenetics, and neuroen- protocol for deriving glucagon-secreting increasingly complex functional models
docrine regulation. a-cells has been established (Rezania of angiogenesis, blood brain barrier, car-
Given these difficulties, how might one et al., 2011). This raises the possibility of diovascular system, nutrient absorption,
begin to systematically tackle the ques- building a human stem-cell-derived islet and liver drug metabolism (Bhatia and
tion of how nutrition affects health? (SC-islet) containing all the pancreatic Ingber, 2014).
Although we cannot literally create a win- endocrine cells types. Many labs are In addition, maturation of the SC-
dow into the human gut to obtain Beau- developing methods to differentiate organs remains a critical challenge for
mont’s view, researchers today would hESCsintoothercelltypessuchasadipo- the success of any stem-cell-derived
benefit from direct access to the human cytes (Cuaranta-Monroy et al., 2014), model. Intestinal SC-gut organoids have
gut. Fortunately new technologies are hepatocytes (Takebe et al., 2013), and improved their function after transplanta-
converging in a manner that may allow myocytes (Salani et al., 2012) and find tion under the kidney capsule of a recip-
us to recreate a functional GI system appropriate culture conditions to keep ient mouse (Watson et al., 2014). It is
in vitro. Our ability to generate organoids cells functional for extended periods of hypothesized that vascularization is crit-
and tissues from human embryonic stem time (Sachs and Clevers, 2014). It is likely ical for this process, so proper maturation
cells (hESCs) or induced pluripotent that we will be able to generate functional of organs may require co-culture with
stem cells (iPSCs) continues to make organoids and cell types such as SC-liver vascular endothelial cells, which can
rapid progress. We may soon be able to and SC-gut for most metabolic organs. also be derived from stem cells (van der
assemblethemintoaminiatureGIsystem The ability to genetically modify stem Meeretal., 2013).
in a dish or even on a chip. This would cells that are used to make the tissues If thesechallengescanbemet,itshould
allow for the manipulation and analysis and organs will be enormously valuable be possible in the future to generate an
of digestion in a reproducible, accurate, in determining which genes function in vitro system composed of several hu-
and large-scale manner, and facilitate in which cells to affect phenotype. Ad- man ES- or iPS-derived cell types and
exploration of the effects of individual vances in gene editing by TALEN and organoids that will absorb food from the
genetic and microbial diversity. Here we CRISPR technologies make it possible luminal side and be connected basally
discuss how these pieces might come to test both loss and gain of function for by fluid to liver, pancreas, muscle, and/
together to aid the science of nutrition. specific genes and organs. or adipose SC-derived tissue, delivering
A complementary advance has come hormones and nutrients from one tissue
Building a Functional from bioengineering, making ‘‘organs- to the next (Figure 1). This would circum-
Gastrointestinal System In Vitro on-a-chip.’’ The chips are microfluidic ventproblemsarisingfromusingimmortal
Intestinal organoids are miniature, three devices, populated by cell cultures orga- cell lines to generate organoids, as these
dimensional, star-shaped versions of the nized and perfused such that they display altered cellular metabolism and
full intestine consisting of a single epithe- resemble tissue and organ biology more generally do not give rise to in vivo 3D
lial layer with crypts and villi. Stem cells than the classic tissue culture in a dish. structures such as a liver lobule or an islet
at the base of the crypt can be isolated The technology aims to preserve the of Langerhans. Clean human primary
from human and mouse primary cultures, advantagesofcellculture,suchasacces- cultures are difficult to obtain, especially
andcangeneratemultiplecelltypes,such sibility, control, and reproducibility in an from many tissues at the same time.
as pit cells, enteroendocrine cells, and environment that recapitulates in vivo In vitro differentiated cells overcome
mucus glands facing a miniature lumen. physiology. these problems and can reproduce spe-
Remarkably, these organoids can be In recent years we have observed a cific genetic backgrounds which can be
grown for long periods of time, and tantalizing increase in the sophistication usefulforpersonalizedmedicineorsimply
have been used by many labs now to of organs-on-a-chip, and an emergence tointroducehumangeneticvariabilityinto
studyinvitro digestive enzymatic activity, of a vibrant bioengineering community research.
development of the gut, bacterial infec- supporting these efforts. We are still Building reliable in vitro systems
tion, and structural aspects of the crypt- learning how to improve upon these first capable of recapitulating digestion is a
villi unit (Sato and Clevers, 2013). Impres- generation models, and there are several monumental task requiring the collab-
sive progress has been recently made challenges to overcome. Manufacturing orative efforts of labs from several fields
toward differentiating hESCs into intesti- is not entirely robust, and requires exper- in biology, chemistry, and engineering.
nal and gastric organoids (McCracken tise of in-house engineers. More impor- Somekeypartsofthesystemaremissing
et al., 2014; Spence et al., 2011) opening tantly, we are missing the functional and integration of the stem-cell-derived
Cell 161, March 26, 2015 ª2015 Elsevier Inc. 13
epithelia(Kimetal.,2012;KimandIngber,
2013). We do not yet know if in vitro sys-
temscanorshouldhostafullcomplement
of gut microbes, how such an ecosystem
can be inoculated and sustained in
culture, or what types of nutrients the
bacteria will require to model digestion
accurately.
In addition, the microbiota responds
dynamically to diet, perhaps more so
than the human gut. It changes with age
and has tight interactions with its host
through metabolites and the immune
system. It is also highly variable between
individuals and societies, representing a
majorsourceofmetabolicvariability(Par-
freyandKnight,2012).Invitromodelingof
the gut microbiome is still at early stages,
and some bacterial strains are not easily
cultured in the lab. Experimenting with
this system will teach us about human-
microbiota interactions and explore how
metabolic variability relates to micro-
biome composition.
Figure 1. Schematic Depiction of a Human Stem-Cell Based in Vitro System to Model In terms of food, the duodenum,
Digestion and Nutrition jejunum, ileum, and colon have different
Food travels through different sections of the SC derived gastrointestinal segments populated by gut digestive functions and encounter food
microbiota.Absorbednutrients,hormones,basallysecretedproteins,andlipidsaretransportedtoanSC- at different digestive stages. In vitro sys-
liver, which recapitulatestoportal-venousaxisinliverlobule.Fluidsarethenfedtoothermetabolicorgans tems should be designed such that
such as SC-islet of Langerhans and SC-WAT. Potentially other SC-tissues can be added. Secreted
hormonesandprotein-lipid complexes are fed back to close the loop (feeding of intestinal segments not hESC-derived intestinal segments form
shownforclarity of presentation). At each point, a fluid or food sample can be drawn, and SC-tissues are separate chambers (Figure 1) that pass
available for biopsies. Important technical consideration such as waste collection and perfusion channels food and chyme at various stages of
are not shown in this schematic depiction.
digestion through the lumen. Tailored
luminal culture conditions and chip me-
tissues in a bioengineered system will certainly not new; however, technological chanics will be required to support the
bechallenging. But considering the prog- advancements in DNA sequencing and introduction of food and provide appro-
ress in the last decade in stem cell other methods have led to a booming in- priate conditions for gut microbiota to
and bioengineering fields, there is good crease in our knowledge on this topic (Li flourish.
reason to believe that creating a physio- et al., 2014). As a result, digestion is now
logical, human-based model of digestion understoodtobeajointhuman-microbial Nutritional Data In Vitro and In Vivo
is possible. process. The food we eat nourishes not Nutrition science is a vast field integrating
only our organs, but also the microbial biochemical, physiological, bioinformatic,
Incorporating Gut Microbes and community that we host. andcultural data. Mathematical modeling
FoodInVitro The challenge of maintaining a micro- and big data analysis are needed, as in
An in vitro human digestive system as bial ecosystem within the lumen of an gut microbiota research, which includes
described above would only partially in vitro system is daunting. We are just sequencing of multi-strain samples,
model digestive physiology. Two other beginning to understand how to culture building statistical models of gut micro-
important factors that need to be gut microbial strains in a dish (Goodman biota, and overlay of metabolic networks
incorporated are food itself and the gut et al., 2011), but initial steps toward this with microbial enzymes. Breakthroughs
microbiota. aimhavebeenmade.Anadvancedmodel in microbiome research have been
The human gut is populated by trillions of a gut-on-a-chip, populated by the gut madepossiblethroughcollaborationsbe-
of bacteria from hundreds or thousands anaerobic bacterium Lactobacillus rham- tween microbiologists, biochemists, and
of strains. Gut bacteria effectively form a nosus GG and human intestinal Caco-2 computational biologists using system
functional organ, composed of many mi- cells, has been developed. Co-culturing biology approaches (Human Microbiome
crobial strains that interact with the hu- bacteria and cells may be easier on a Project Consortium, 2012).
man host and perform various metabolic chip due to the constant flow of nutrients Metabolomics, the high-throughput
functions. The recognition of the impor- and differential culture conditions on the mass spectrometry quantification of me-
tance of microbiota-host interactions is luminal and apical side of the intestinal tabolites in cells, organs, and body fluids,
14 Cell 161, March 26, 2015 ª2015 Elsevier Inc.
benefits from the collaborative efforts patient to the prescribed diet, effects An in vitro system has applications for
of biochemists and computational biolo- associated with the immune and nervous screens of food additives or synbiotics.
gists,andthedataanalysistoolsarecom- systemsofthemodelorganism,andother Currently, studies are done on cell lines,
parable to those of sequencing and gene uncontrolled variables. using mice or cohorts of humans, and
expression (Melamud et al., 2010). In the Liveimagingismucheasierinvitrothan are not practical for the challenge. For
future, many more studies will likely use in vivo. Imaging metabolic or adaptive example,howwouldwetestwhichbacte-
metabolite profiling to describe the phys- processes is limited today to simple rial strains are most efficient at digestion
iology of human subjects in health and in vitro cultures, or to snapshots from of complex carbohydrates? Numerous
disease, leading to discovery of novel in vivo models.UsinghESCreporterlines, combinations of bacterial strains should
roles for metabolites and refining the one will be able to track the expression be tested in several dietary conditions
known metabolic networks at the cell of specific genes, cell composition, and human genetic backgrounds. Such
and organism-microbiota levels (Ryan secretion of factors, and changes in the experiments would require thousands
et al., 2014). morphology of human tissues. of mice, but an in vitro system might
Similarly, nutrigenetics and nutrige- In all, the inherent complexity of nutri- be scaled up for the task. Similarly, a
nomics advance our understanding of tion and digestion may be reduced and controlled system is ideal for studies on
human metabolic variability using human dissectedwithahESC-basedinvitrosys- the uptake of food and drugs. The differ-
genetics and microbiome sequencing. tem. Access to high quality imaging, me- encesbetweenmodelorganismsandhu-
A classic example is the discovery that tabolomics, and genomics data will allow mans become crucial in studying drug
mutations in the ldlr gene cause familial analysisoftractablemathematicalmodels metabolism, and an in vitro system might
hyperlipidemia. More recent efforts to basedonlargedataandtestingofspecific better model drug metabolism, improving
relate genetics, diet, and disease include biologicalhypotheseswithouttheinherent the efficiency of drug development.
a series of biochemical and genome- difficulties of immortal cell lines. We envision through this system will
wide association studies aimed at address many puzzles in the physiology
exploring connections between genetic Applications and Limitations of a ofdigestion.Wewillbeabletowatchbac-
polymorphisms, red meat, and vegetable HumanStem-Cell-BasedSystem teria break down food and pass meta-
consumptiontothedevelopmentofcolo- Aninvitro, complexstem-cell-basedsys- bolites to enterocytes, measure the level
rectalcancer(Figueiredoetal.,2014).The tem can fill the gap between an animal of hormones as they are secreted, and
Segal and Elinav labs take a different in vivo model and cell lines, but cannot watch the intestine and microbiota adapt
approach to understanding human meta- modelthefullcomplexityofaninvivosce- to a change in diet. It should be possible
bolic variability by measuring glucose nario and is not as straightforward as a tomodelthedigestivetrackfollowingbar-
levels and food intake over the course of cell line or primary culture. However, it iatric surgeries and study the positive and
a week and sequencing the gut micro- can complement existing models and negativeeffectsofsurgeryonmetabolism
biome of individuals. The results are fed humanstudies and create an opportunity (MingroneandCastagneto-Gissey,2009).
to a machine learning algorithm that to better address physiological and nutri- It will also permit modeling of fecal trans-
generates a personalized diet aimed at tional questions. plants in specific genetic backgrounds
reducing glucose spikes following meals The ability to use iPSCs and genome and diseases.
(www.personalnutrition.org). editing technology has been already put Ultimately, our hope is that this in vitro
Manytoolsandapproachesdeveloped to effective use for disease modeling system will go beyond the physiology of
today will be instrumental for analyzing (Rashid et al., 2010; Wang et al., 2014). digestion into the field of nutrition. The
thehESCbasedinvitrometabolicsystem A stem-cell-based system can be used aforementioned unanswered questions
weenvision.Thevariousparametersinflu- to study nutritionally related diseases. in nutrition involve the long term effects
encingmetabolismcanbewellcontrolled For example, several genes have been of diet, but currently organs-on-chips
and,comparedtoinvivostudies,itshould associated with intestinal inflammatory have been limited to shorter time scales
be possible to collect high quality, repro- diseases, but the role of only a handful on the order of weeks. This limitation
ducible data. For example, we will be of genes has been elucidated (Murthy makes such systems less valuable for
abletosamplefluidscontainingabsorbed et al., 2014). It is not known whether the study of long term processes such
metabolites from different positions in changesintheimmunesystem,theintes- as chronic disease development. How-
the in vitro gut and understand how they tine at large, or gut microbiota triggers ever,if indeed theinvitrosystemcansus-
are affected by gut microbiota, a sudden disease in genetically susceptible pa- tain cultures for longer periods of time,
change in diet, antibiotics, or fasting and tients. Moregenerally,theabilitytogenet- one could follow the metabolic profile of
feeding. Biopsies from the in vitro system ically modify stem cells that are used to tissues following long term exposure to
could teach us about the proteomic make tissues and organs will be enor- a specific diet. One could capture the
and transcriptomic profile of the liver in mously valuable in determining which development of insulin resistance in a
response to feeding, with high temporal genes function in which cells to affect tissue as a function of diet in a system
resolution. Alternatively, biopsies and phenotype.Theadvancesingenemodifi- simple enough to isolate in vivo effects
fluid samples may be obtained from the cation in this regard are all in place, mak- exerted by the immune and nervous sys-
systemoveralongperiodoftime,without ingit possibletotestbothlossandgainof tem, but complex enough to capture mi-
worrying about the compliance of the function for specific genes. cro-environmental effects, such as liver
Cell 161, March 26, 2015 ª2015 Elsevier Inc. 15
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