Abstract
When an oocyte is fertilized, it divides and – within days – stem cells arise. These embryonic stem cells (ESCs) build the entire body with all its organs and specialized cells. Consequently, they are said to be ‘pluripotent’. Once the body plan is established, the individual organs are maintained and repaired lifelong by much more specialized stem cells, dedicated to the organ in which they reside. These are termed tissue stem cells or adult stem cells (ASCs). Since ASCs produce only a limited number cell types, they are termed ‘multipotent’. Together, these two stem cell types hold promise for eternal life in the (probably distant) future. Meanwhile stem cells are rapidly changing the face of biomedical science and are actively being applied in the clinic. Below, I discuss principles of stem cell biology and of the mini-organs that can be grown from stem cells outside the human body, in the lab.
It was in World War II when stem cells came to prominence. Radiation sickness was first described by the Red Cross Hospital Surgeon Terufumi Sasaki when nuclear bombs were dropped on Hiroshima and Nagasaki. Dr Nagasaki noted the gradual decrease of white blood cells from the blood of patients within weeks after the exposure to radiation. Subsequent observations in the 1950s revealed that bone marrow cells reversed the loss of blood cells when injected intravenously into radiated recipients. The conclusion from these experiments was simple and powerful: rare but powerful cells exist within the bone marrow that protect against the consequences of radiation. These cells produce all known varieties of blood cells: platelets, white cells and red cells. Moreover, they can recreate themselves, a phenomenon called self-renewal. Inspired by these findings, Thomas and coworkers endeavored to develop the transplantation of these bone marrow stem cells for therapeutic purposes. Realizing that it would not be straightforward to transplant cells between unrelated individuals, their first attempt involved identical twins, one of whom suffered from leukemia and received bone marrow from the healthy sibling.
Bone marrow transplantation then rapidly developed further. An important step involved the replacement of a ‘qualitative’ phenomenon – the rescue of blood cell production by bone marrow from a donor – by a much more defined procedure. For this, the exact identity of the elusive multipotent hematopoietic stem cell (HSC) needed to be established.
For this, the Toronto-based stem cell scientists Till and McCulloch devised a simple, animal-based lab test allowing to visualize HSCs. This so-called spleen focus-forming assay proved that individual bone marrow cells could indeed generate large numbers of all other blood cell types. Combined with a series of in vitro technologies that were developed later, the definitive identification of clonogenic HSCs was accomplished which heralded the start of the still sprawling HSC discipline.
Sixty years beyond these breakthrough discoveries, bone marrow- and cord blood-derived HSC transplantation now represents one of the routine therapeutic modalities used for malignant disease in hematology, but also for certain autoimmune diseases and even for multiple sclerosis.
From decades of studies on HSCs, a generally accepted definition of HSC attributes and of the architecture of the differentiation hierarchy driven by HSCs has arisen.
1) A key definition of an HSC involves the potential to recreate itself, i.e. to self-renew. This attribute is termed longevity; 2) A single HSC can produce all blood cell types. This attribute is termed multipotency. Direct daughters of HSCs are proliferative and while they increase their numbers, they gradually specialize into one of the different cell type lineages: platelets, red blood cells, lymphocytes, monocytes, granulocytes. This process involves a highly controlled stepwise choreography and is irreversible.
In the past few decades, new technologies have allowed the identification of stem cells in a variety of solid organs and tissues. These tissues can crudely be classified into two groups. In most organs (such as prostate, lung, or liver), cells do not divide much – if at all – under normal conditions. Yet, these organs can display vigorous waves of cell division when damaged. Other organs (such as the skin, or the inner lining of esophagus, stomach and gut) show constant cell division. This results in the continuous replacement of the pertinent tissues by healthy, young cells. The bone marrow and the blood cells that are generated from HSCs belong to this second class of tissues.
Research in our lab has focused on the cells that cover the insides of the small bowel. The tissue that they form is called the intestinal epithelium. While studying the intestinal epithelium, we have come to realize that intestinal ASCs display a series of highly unexpected attributes, some of which also define ASCs in other solid organs. The small bowel epithelium is highly compartmentalized in so-called crypt-villus units. Mouse guts comprise about one million of such units while the human small intestine might contain some one billion of these. A villus protrudes from the wall of the short bowel into the central ‘channel’ or lumen; this architecture dramatically increases the capacity of the intestinal lining to absorb nutrients from the gut lumen. The villus surface consists of a single layer of specialized cells that perform various functions to digest food and absorb nutrients, while keeping unwanted entities such as indigestible food components, bacteria etc., outside the body proper.
Within a villus, blood vessels allow the further transit of nutrients towards the liver. Surrounding the villus base, 8-10 small pits protrude outward into the wall of the gut. These pits were first discovered by a young German scientist, Jonathan Nathanael Lieberkühn (1711-1756), in Leiden in the Netherlands (Fig. 1).
They have since been named crypts of Lieberkühn. For his studies, he injected heated wax into blood vessels of isolated organs to visualize tissue architecture. In the middle of the last century, it was realized that crypts are the site where the most active stem cells of the mammalian body reside. These intestinal stem cells drive a stem cell hierarchy which populates the remainder of the crypt as well as the surface of the villi. Six main cell types can be distinguished (Fig. 2).
Enterocytes represent the most common cell type on the villus and are responsible for the absorption of nutrients and liquids. For this, it carries a large number of tiny folds on its surface, together forming the so-called brush border, again to maximize nutrient absorption. Paneth cells are located at the base of crypts; their main function involves the defense against luminal microbes.
They perform this function through the production and secretion of bactericidal peptides and proteins. The function of Tuft cells remains somewhat elusive but they appear to play a key role in immunity against helminths. Goblet cells are secretory cells which produce mucus to enable smooth transport of the food bolus through the gut lumen and to restrict entry of microbes into the host body. Enteroendocrine cells come in five or six flavors, each producing a unique set of hormones, which control many aspects of metabolism, hunger and satiety.
Lastly, Microfold (M) cells only occur in the intestinal lining that covers specialized lymphoid structure that are termed Peyer’s patches. M cells transport small and large antigenic particles from the gut lumen to the underlying lymphoid cells and thus play a key role in establishment and maintenance of mucosal immunity.
Leblond (Fig. 3) and Stevens were the first to describe the kinetics of the physiological behavior of the stem cell hierarchy of the intestinal crypt-villus stem cell units. Their landmark study was published in 1947 and was performed on rats. They observed that adult rats constitutively generate new cells in great numbers in their crypts. They also claimed – to the disbelief of many – that the lifespan of a single intestinal epithelial cell would not be much more than a few days. They acknowledged also the consequence of this observation: the dramatic, daily production of cells in crypts had to be in cue with a location elsewhere in the crypt-villus units where the cells – after having enjoyed a brief lifespan – would meet their demise. Leblond and Stevens thus wrote, “…the cells formed in the crypts of Lieberkuhn move upward along the side of the villi to be ejected when they reach the villi tips”. An immediate conclusion from these notions would be that the intestinal stem cells that drive the vigorous French-Canadian stem cell pioneer’s cell replacement would live at or near the base of the crypts.
In contrast to all other cells of the intestinal epithelium, the stem cells would be defined by two key attributes: they should continuously regenerate themselves (longevity) and they should produce all other cells of the tissue (multipotency).
Formulated more directly: As a laboratory mouse lives about three years, crypt stem cells should be able to persist for three years, and during that time should continuously generate enterocytes, goblet cells, Paneth cells, enteroendocrine cells and M cells. J.P. Leblond initiated the research into the identity of the stem cells of intestinal crypts. Leblond and Cheng first made a key observation: Paneth cells are not the only cell type present at the bottom of the crypt. Intermingled between the large, non-dividing Paneth cells with their eye-catching granules, careful examination using electron-microscopic techniques unearthed the existence of a tiny cell type, consisting of little more than a nucleus and a few organels.
These cells turned out to divide every day for the lifetime of the mouse and based on their columnar morphology, they were termed crypt base columnar (CBC) cells. Joseph Paneth also noted these cells in his study from 1887 in which he described the Paneth cells (Fig. 4).
Much more recently, Nick Barker and others in my lab identified Lgr5 as an exclusive molecular flag, present uniquely in CBC cells. Nick Barker went on to create a number of knock-in mice targeting the Lgr5-locus (Fig. 4). Using these mice lines, we confirmed all essential predictions made originally by Leblond: The CBC cell, which uniquely expresses Lgr5, is the crypt stem cell. It continuously divides and does so lifelong. It generates all other cell types of the epithelium. Paneth cells are a key part of the stem cell niche which supports the vigorous activity of the CBC stem cell. Non-stem cells display plasticity and can dedifferentiate to become CBC cells, when the original CBC cells are lost.
In the early 2000s it was widely believed that ASCs could not be maintained outside the human or mouse body for more than a few days, let alone that ASCs could be encouraged to increase their numbers in a Petri dish. It is still true that after almost seven decades of the clinical application of bone marrow transplantation, all attempts to amplify HSC numbers in vitro have remained futile. The advantages of such stem cell expansion in vitro are obvious. Currently, it takes HSCs isolated from one donor to treat one patient. If stem cell numbers could be boosted in vitro, multiple patients could be treated with a stem cell isolate obtained from a single donor, or a patient could be treated multiple times using a single stem cell sample. Based on our observation that CBC cells undergo one cell cycle each day, CBCs have gone through one thousand consecutive cell cycles in the gut of an aged lab mouse. We had previously determined which growth signals are key to maintain active CBC cells in a mouse in vivo. Based on these observations, Toshiro Sato in our lab designed a 3-dimensional culture system for CBC cells with the intention to amplify their numbers in vitro. The approach is based on a hydrogel consisting of collagen and Laminin (Basement Membrane Extract, or MatrigelR). To the hydrogel, the Wnt agonist R-spondin1 is added. We later discovered that R-spondins are ligands of the 7-transmembrane Lgr5 receptor. Two other key growth factors are Epithelial Growth factor and Noggin, a BMP-blocking secreted protein. When single CBC cells, sorted from Lgr5-GFP mice, are placed in this hydrogel-based medium, defined structures grew out, rather than the expected lumps of CBC stem cells. Careful analysis of these structures revealed that they contained all cell types of the gut epithelium, in normal ratios and at their normal location: Paneth cells and CBC stem cells at the base of the protruding crypts and all other cell types lining the central lumen. Dr Toshi Sato named these ever-expanding structures “mini-guts”. A more scientific term is ‘small intestinal epithelial organoids’. The term ‘organoid’ is now broadly used for structures that are grown from stem cells and that recapitulate key features of the organ of interest in terms of architecture, cell type composition and function.
Thus, “mini-guts” are grown in vitro from a single Lgr5 stem. They faithfully phenocopy central aspects of the physiology of normal gut epithelium. Bud structures that emanate from the periphery of organoids contain resident CBC cells, Paneth cells and rapidly-dividing transit-amplifying cells. These crypt-like buds create a flow of differentiated cells of the various lineages towards the lining of the central lumen. The dynamics of this process closely mimics that of the crypt-villus units of the small intestine: CBC cells generate daughter cells each day. These daughter cells themselves proliferate for a while, after which they mature into any of the prototypic cell types and after some days die and are discarded into the luminal space of the organoids.
A large batch of mini-guts was grown from a single adult colonic CBC cell, isolated from the Lgr5-GFP transgenic mouse line. The resulting organoids were transplanted in the lab of our collaborator Mamoru Watanabe. They were introduced intraluminally into the inflamed colons of several dozen mice treated with the chemical DSS to induce a colitis-like syndrome. The transplanted mini-gut organoids integrated fully into the damaged wall of the DSS-treated colons and maintained functionality for the duration of the experiment. Based on these and subsequent experiments, Watanabe and colleagues have embarked on a project to treat treatment-resistant inflammatory bowel disease patients with autologous colon epithelial organoids.
Since these initial mini-gut studies, we and many others have developed protocols to grow organoids from numerous other mouse and human organs (Fig. 5).
These protocols invariably allow the establishment of organoids that capture key characteristics of the organ of interest. Adult stem cell-based organoids simply require a small piece of tissue, obtained from a biopsy or from a surgical resection sample as starting material. A parallel technology starting from pluripotent stem cells was originally pioneered by Dr Yoshiki Sasai, who focused on generating structures of the central nervous system and the retina. ASC-related organoid approaches exploit the maintenance and repair capacity of adult stem cells, which are fully fated towards the organ in which they reside. By contrast, pluripotent stem cell-derived organoids exploit the capacity of ES cells or iPS cells to generate each part of a mammalian body; the organoids are fated in vitro towards the organ of interest by mimicking the developmental journey they would have experienced in the developing embryo. A multitude of technologies, discoveries and applications have emerged around this organoid concept. Breathtaking types of organoids derived from iPS cells are the cerebral organoids (or “mini-brains” of Lancaster and Knoblich), and the mini-kidneys of Melissa Little and colleagues from Melbourne.
Organoids representing human tissues are increasingly embraced by basic biomedical scientists. Organoids derived from healthy human tissues find applications as alternatives to animal experimentation. Human organoids may also better represent diseases: when directly grown from cancers, they appear superior models of human cancer when compared to ‘classical’ cancer cell lines. Organoids also find applications in other disease areas: In infectiology, organoids allow the study of a variety of pathogens, be it bacteria such as the stomach ulcer-causing Helicobacter; viruses such Noro- and influenza viruses, RSV and the SARS-CoV2 virus, and eukaryotic microbes such as Cryptosporidium.
Organoids model hereditary diseases such as cystic fibrosis. In oncology, organoids can be directly grown from tumors and they are now believed to faithfully recapitulate tumor cells of individual patients.
Organoids are being validated for personalized medicine strategies. Tissue samples taken from patients can be grown as organoids to serve as avatars of the pertinent patient. Drug testing of the avatar-organoid predicts and allows tailoring of drug- or radiation-based treatment of individual patients. Indeed, organoid approaches already allow rapid ex vivo testing of drug responses on tissue samples obtained from individual patients. As a preeminent case study, a minigut-based cystic fibrosis (CF) assay yields a test result within 14 days after obtaining a small biopsy from a given CF individual. The outcome of this test is unambiguous in predicting if the patient will respond clinically to the CF medicine. In the Netherlands, the CF organoid test has been performed for a majority of CF patients. When a positive assay result is obtained, the corresponding CF individual will be prescribed the (expensive) CF medicine. Organoids offer a similar opportunity to cancer patients (see above). A number of clinical studies has already underscored the high predictive value of cancer organoid-based drug sensitivity screening in a personalized health care setting.
Cancer organoid-based assays allow evaluation of multiple therapeutic regimens prior to selecting the most optimal one to be given to the patient. It will however still take some years before this approached is validated well enough to become a routine diagnostic tool. For that, organoid derivation and drug screening will require automation and a significant improvement in terms of speed and cost.
References: This manuscript summarizes a lecture given at Vatican City in 2022. It was based on several reviews written by the author.
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