Stem cells are a group of cells inside an organism that is yet to be differentiated into mature functional cells but have the capabilities to differentiate into any kind of cells, alongside being self-renewal (1). These abilities in differentiation into any kind of cells and self-renewal of stem cells prove to be important in developing and regenerating certain cells in the body. Stem cells are grouped by highest differentiation ability, in descending order: totipotent, pluripotent, multipotent, oligopotent and, lastly, unipotent (2), which is from being able to generate an entire organism and extraembryonic tissues, to only a single lineage of cells.

Totipotent cells are stem cells that can differentiate into all types of cells in an organism, together with the development of extraembryonic tissues such as the placenta (3). Pluripotent cells are stem cells that can differentiate into any three distinct germ layers: endoderm, mesoderm, or ectoderm (4). Each layer is responsible for specific organogenesis, such as the endoderm layer that develops the gastrointestinal tract, liver, pancreas, the mesoderm layer into the circulatory system and the musculoskeletal system, and the ectoderm responsible for hair, skin, nails, and nervous system (5). Multipotent stem cells are also known as adult stem cells or somatic stem cells. Examples of multipotent cells in the human body include but are not limited to, hematopoietic stem cells, mesenchymal stem cells, skeletal stem cells, neural stem cells (6), and intestinal stem cells (7). Similarly, oligopotent and unipotent stem cells also contribute to their tissue-specific cell lineages, capable of differentiation into a limited number of cells or terminally differentiated (1).

Continuing from the multipotent stem cells, examples of stem cells with this differentiation ability are intestinal stem cells (ISC), which are undifferentiated stem cells located in the crypts of intestinal epithelium (8). These stem cells are the cells that give rise to the intestinal epithelium with its variety of cells and many more functionalities by continuously proliferating and dividing (9). From the ISCs, the progenitor cells continue to divide and differentiate into specific cell lineages such as absorptive or secretory cells (10). Facilitation of these processes is crucial to balance both the proliferation of new cells and the removal of old or damaged cells, which is provided by the plethora of appropriate biochemical signaling pathways. Without it, DNA mutations can especially accumulate in cells, leading to uncontrolled differentiation and potentially producing harmful cancerous cells (11).

The gut microbiota refers to a dynamic community of microorganisms that live in the human gastrointestinal tract (12). These consist of various bacterial species, viruses, archaea, and fungi. As the gastrointestinal tract, particularly the stomach, small intestine, and large intestine, are responsible for the digestion and absorption of food and drinks ingested, this provides a perfect environment for microorganisms to colonize the site of the nutrients and safety provided by the human host (13, 14). Despite the enormous number of microorganisms in the gastrointestinal tract (GI), a large proportion of them have a symbiotic relationship with the human host through many biological processes. For example, the gut microbiota helps mediate body homeostasis, strengthen the integrity of the gastrointestinal tract, and decrease the colonization of foreign or pathogenic strains of microorganisms in the GI tract (15). Furthermore, the gut microbiota regulates the innate and adaptive components of the immune system such as antigen-presenting cells, regulatory T cells, and innate lymphoid cells (16).

Unlike any other stem cells, intestinal stem cells exist in tandem with the indigenous gut microbiota population in the GI tract, albeit living separately within their integrity and maturation of the intestinal epithelium depend on the harmonization of both the intestinal stem cells and the gut microbiota working together. The absence of either will disrupt the equilibrium between the two, either with dysbiosis, the imbalance of the gut microbiota, or inflammatory bowel disease (17).

Hence, this review aims to address the relationship between ISC and gut microbiota within the context of regenerative medicine. The characteristics of ISC are explained thoroughly by investigating the Lgr5 and + 4 stem multipotent intestinal stem cells that all intestinal epithelial cells are derived from. The diverse bacterial and fungal community that makes up the gut microbiota in the human gastrointestinal tract is also investigated for the roles they play in the host organism. The relationship between the ISC and the gut microbiota is then further examined to determine their combined roles in regulating the homeostatic function of the intestine. A clinical trial is also included on ISC and gut microbiota to highlight these specific research studies as summarized in (Figure 1). Future perspectives and recommendations are also discussed on progress in the field of study.

The intestines have been able to perform their function of digestion and absorption of nutrients for the body due to the meticulous architecture of various cells, substances, and their microenvironment. Both the small and the large intestine are composed of a single layer of columnar cells that line the epithelium of both organs. The small intestine also has villi protruding into the lumen and Lieberkühn crypts, while the colon also has the crypts and absorptive plates which are alternatives to villi, with these features to increase the total surface area of cells that touch the food ingested (18). These crypts house intestinal stem cells (ISCs), which all the intestinal epithelial cells originate from (9).

Intestinal epithelial cells, also known as mature intestinal cells, consist of two lineages: the absorptive lineage and the secretory lineage (10). The absorptive lineage with enterocytes, whose main function is to absorb nutrients from food, and M cells or microfold cells are to continuously examine intestinal microbes and mediate the appropriate mucosal immune response (8, 19). Secretory lineage cells comprise four types (i) enteroendocrine for hormone production and release, (ii) Goblet cells for mucus production and secretion, (iii) opioid-secreting Tuft cells (20) and (iv) multifunctional Paneth cells for the secretion of antimicrobial peptides as well as signaling molecules to facilitate the maturation of intestinal stem cells and their subsequent progenitor cells (9, 21).

As mentioned above, mature epithelial cells are derived from multipotent intestinal stem cells that reside in the crypt of the intestinal epithelium. The turnover of intestinal epithelial cells is maintained by differentiation of ISCs into different cell lines of secretory or absorption by maturing first into the respective progenitor cells (22, 23); also known as transit amplifying cells (TA cells). These secretory or absorptive progenitor cells can be further committed to their respective lineage and differentiate into secretory Paneth cells or absorptive enterocytes, respectively (24). ISCs, TA cells, and other types of absorptive and secretory cells are located in the single layer of the intestinal epithelium. All except ISCs and Paneth cells continuously move across the crypts and villi and toward the lumen before ultimately shedding into the lumen (25).

The intestinal stem cells were first discovered to be located in between Paneth cells at the bottom of a mouse intestinal crypt in alternating patterns. These cells were coined as crypt-base columnar cells (CBCs) and demonstrated their stemness, which is the ability of the cells to proliferate and differentiate. This can be confirmed by the fact of a similar radioactive component that was observed in intestinal cells that differentiated from the CBCs (26). The gene Lgr5+ was discovered as one of the genes specific to CBCs (27) during the investigation of the Wnt signaling pathway as an important homeostatic pathway in intestinal regeneration. These LGR5-expressing cells were collected from intestinal crypts and continuously self-renew and were also capable of differentiating into the two main intestinal cell lines and are long-lived under conditions enhanced by specific growth factors (20, 28, 29). Thus, the self-renewal and the ability to differentiate into multiple lineage-specific progenitor cells meet the criteria of stemness as described in (Figure 2).

Besides Lgr5+ stem cells, another distinct type of intestinal stem cell called +4 stem cells are found in the ISC. The name of the +4 stem cells derives from the position of this cell type, as they are the ‘fourth’ from the bottom of the crypt (30). These +4 stem cells are observed to be more quiescent than Lgr5 ISCs and were generally regarded reserve stem cells that help replenish the actively used Lgr5 stem (31), either by generating intestinal progenitor cells (32) or by dedifferentiating transit-amplifying cells back into Lgr5 stem cells with the transcription factor ASCL (33). It has been observed that the Lgr5 stem cell pool can be replenished by both absorptive and secretory progenitor cells, and mature Paneth cells were also found to be capable of de-differentiating (34–36).

The gut microbiota refers to the abundant and complex collection of complex microorganisms that reside in the gastrointestinal tract. These consist of a diverse group of bacteria, viruses, archaea, and fungi, which can reach a staggering number of 100 trillion microbes when accumulated (14). As the GI tract, particularly the stomach, small intestine, and large intestine, are responsible for the digestion and absorption of ingested food and drinks, this provides a perfect environment for microorganisms to colonize the site for the nutrients and safety provided by the host (13, 14).

It is important to note that the gut microbial community is enormous and diverse; therefore, a single microbiota composition is not properly optimized, as it is unique individually. These depend on many factors such as early life factors such as type of delivery, milk feeding from the mother and weaning period, as well as external factors such as frequency of exercise, sociocultural diet habits, BMI level, and enterotypes (12).

Besides, variations in the gut microbiota intra individually can also be affected such as in certain anatomical regions. For example, the short transit time of the intestine of 3 to 5 h and the high concentrations of bile in the small intestine prove to be a difficult environment for microbes to thrive. Thereby, a study has revealed that facultative anaerobes such as gram-positive lactobacilli, enterococci, and streptococci, as well as gram-negative Bacteroides and Proteobacteria, were observed through molecular analysis to populate the jejunum and ileum region (65, 66). On the contrary, the neutral to mildly acidic pH and the slow flow rate of the large intestine harbors a more diverse gut microbiota, mainly consisting of obligate anaerobes. In healthy individuals, there is minimal microorganism colonization on the surfaces of the large intestinal epithelium and an inner mucin layer. The distal mucin layer has bacteria with special mucin-degrading properties, such as Akkermansia muciniphila and Bacteroides spp., which are part of the normal microbial flora, the former having immunomodulatory responses in mouse models (67, 68).

Primarily, small metabolites that are produced from their metabolism are the main essential method in which the gut microbiota can indirectly influence the intestinal epithelial layer function and development. Direct exposure of ISC to gut microbes, indigenous or not, can be devastating considering the vital role of ISC and progenitor cells’ long-term integrity and functionality. Therefore, the main method of the effect of the gut microbiota on ISC is mediated by their interactions with intestinal epithelial cells in the upper epithelium layer, which will subsequently cause the appropriate cascade of responses in various feedback systems that can protect ISC and progenitor cells from biological damage or promote regeneration of the epithelial layer through differentiation and proliferation. Despite the enormous number of microorganisms in the gastrointestinal tract, a large proportion of them have a symbiotic relationship with the human host through many biological processes (15). This includes, but is not limited to, the defense against pathogenic microbes, the immunomodulatory effects of sending signals to host immune cells and helping in host nutrient metabolism (69).

Inquiries regarding relevant clinical trials are conducted in the clinicaltrials.gov database, and there are more than 150 clinical trials that are related to the either the topics of stem cells, gut microbiota or both. Upon various filtrations, currently there is only one completed clinical trial that specifically argues regarding ISC and gut microbiota. The clinical trial is mainly to investigate the relationship between ISCs and diseases related to gastrointestinal tracts. The study by Helse Fonna focuses on investigating the effects of symptoms, quality of life, fatigue, change in intestinal stem cells, enteroendocrine cells, immune system, and dysbiosis before and after fecal microbiota transplantation (FMT). FMT refers to the transfer of the intestinal microbiota of the fecal material of a healthy individual suspended in liquid form to the gastrointestinal tract of another person (114). It is the most widely used therapeutic procedure for people who suffer from an infection of the Clostridium difficile bacteria, which may be present when there is an imbalance in the normal gut microbiota due to repeated antibiotic treatment (114, 115). Although the exact procedures may differ from various medical institutions, the general principle remains the same in all FMT (116). Other clinical trials are correlated with FMT, but these clinical trials do not investigate the effect or relation with ISC, therefore, these clinical trials are excluded in this manuscript. Additionally, certain clinical trials are focused on the usage of other types of stem cells such as hematopoietic and peripheral blood stem cells, which are not relevant and briefly listed in Table 4.

Briefly, the protocol of this relevant clinical trial is carried out first by collecting the stool sample from a healthy individual and then rigorously and subsequently screened for transmissible diseases, blood and genetic screenings for specific intestinal parasites, C. difficile, hepatitis A-B-C, syphilis, HIV, and any other diseases chosen by local protocols (117). The screened stool sample is then diluted with PBS or other saline before the solution is homogenized and filtered to remove any solid particulates. The resulting solution is then either frozen for long-term storage or applied directly into the recipients’ intestinal tracts via colonoscopy (117). The clinical trial is also conducted in a double-blind manner to prevent discrimination and the participants were also randomly assigned into three distinct interventional groups with a placebo group (patients receiving their own fecal matter), 30 g of healthy donor fecal matter group, and 60 g of healthy donor feces. The outcome is then assessed by having the participants to answer certain questionnaires regarding their global improvement in irritable bowel syndrome symptoms, fatigue, and quality of life, all within the time frame of 3 months. However, no results have been published for this clinical trial.

The gastrointestinal tract has gone through countless epithelial turnovers and regeneration in its lifetime, but more substantial research regarding the exact mechanisms by which the proliferation and differentiation of ISCs and gut microbiota with its metabolites are interacting with each other indirectly remain to be done. The critical roles of both ISCs and gut microbiota are to maintain the normal functioning of the gastrointestinal tract, and without these two important players, the gastrointestinal tract alone would not be able to perform its crucial role in the absorption of nutrients.

Both intestinal stem cells and the gut microbiota have their own set of mechanisms that work in complement to each other illustrated in Figure 3. Evidence has shown some of the mechanisms were agreed upon by multiple researchers, for example, bacteria in the GI tract may provide essential substances such as SCFA that can act as additional energy sources for intestinal epithelial cells, and in response, ISC can differentiate into Paneth cells that secrete antimicrobial chemical substances and M cells that continuously sample any microbes in the GI tract, both serve to protect the indigenous gut microbiota from being eliminated by foreign microorganisms. Conversely, the absence or imbalance in the normal gut microbiota known as dysbiosis has been observed to have many derogatory effects in the host such as C. difficile infection that prevents the recovery and regeneration process of the intestinal epithelial, irregular hyperplastic intestinal crypts to be observed in the GI tract without essential tryptophan metabolizing bacteria to name a few (Figure 4).

Due to the microscopic size of cells and bacteria, as well as the hundreds of microbes; it is a daunting challenge to pin down every single relationship and connection of the ISCs and gut microbiota in the proper functioning of the gastrointestinal tracts, particularly of the intestines. Much has yet to be discovered. Several studies mentioned in this review involved laboratory animals or intestinal biopsies to explore ISC, gut microbiota, or both at once by inducing experimental variables in the test subjects, and subsequent results are observed. Variables that could affect the research outcome are diet diversity, the number of processed foods, and newly approved food additives in the daily diet, all of which can cause dynamic changes in an individual’s gut microbiota.

In the past, culturing bacteria commonly found in the human gastrointestinal tract has been a great challenge for biomedical scientists to taxonomically collect and classify the human gut microbiota (118, 119). Some of the bacterial species are very minuscule in number or regarded as unculturable, which can be attributed to the extreme differences in the microenvironment of inside and outside of the human gastrointestinal tract. A study by Ito et al. has successfully cultured a part of this otherwise ‘unculturable’ bacteria population using specific culture media such as chocolate agar, DHL agar, and gut microbiota medium, among others (120). However, there are still many intestinal bacteria with unknown functions that cannot be identified with conventional culturing methods.

The current technology of the next-generation sequencing (NGS) allows the identification of the genus and species of certain bacteria without depending on the lab culturing. At present, the commonly adapted NGS methodologies for sequencing bacterial DNA or RNA are shotgun metagenomic sequencing and amplicon sequencing (121). The shotgun metagenomic sequencing method uses the attachment of adapters and barcodes to fragmented bacterial DNA segments in a randomized manner. The added DNA segments are then cross-referenced with reference databases such as Genbank and Reference Sequence. Amplicon sequencing, also known as 16 s rRNA sequencing method instead amplifies a particular segment of the DNA and determines the nucleic acid sequence of the amplified product. This is always done on the 16S ribosomal RNA gene for the highly preserved sequence among bacteria and having nine hypervariable regions that are unique to each bacterial genus, thus allowing identification of the said unculturable bacteria (121).

The NGS method offers many benefits when it is applied in the clinical microbiology field. This includes a reduction in the turnaround time for the release of patient test results by the attending physicians, a wide selection of identifiable bacterial or fungal etiological agents, and less hands-on involvement of laboratory technologists, especially with slow-growing or fastidious bacteria (122). Rapid and accurate diagnosis is essential in treating patients. Approximately 60% of acute encephalitis cases have been undiagnosed, possibly due to the lack of specific assays that can detect more than 100 etiological agents of encephalitis (123). The same can be applied to other pathogenic organisms, where the NGS method can rapidly identify the presence of these organisms as opposed to individual assay kits to detect them (124). Therefore, the use of the NGS method can offer a great advantage in discovering novel microbes whose unknown or additional roles in the gastrointestinal tract can be discovered and investigated for their cellular activities.

Additionally, the gut microbiota has many essential roles in the maintenance of a properly functioning gastrointestinal tract, particularly in its anti-cancer properties. As demonstrated in chapters 2 and 3, there are many intertwining feedback systems and regulatory pathways with both the ISC and gut microbiota on the homeostasis of the intestinal epithelium and the absence of one can affect the other, which may finally result in disorganized intestinal crypts and reduced proliferation and turnover rate of the epithelium, thus impeding the intestines of its function. This leads to an interesting connection with another part of the body where there is also an indigenous microbial flora that resides outside the cells, and without this microbial community, it can subsequently affect the host and also its dependencies. An example of such a site is the human breast and its indigenous microbiota (125). Nevertheless, more studies are warranted to explore more interactions between ISC and gut microbiota, and the techniques that have been used when exploring their intricate relationships can be applied when novel studies are done on other bodily sites.

FN, GT, WW, and Y-FT: conceptualization and visualization and supervision. GT, WW, and Y-FT: validation. ANAS: writing-original draft preparation. ANAS and ASJ: drawing of figures. GT, WW, FN, Y-FT, and ASJ: writing-review and editing. FN: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

This review is prepared as part of the collaborative work funded by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (FRGS) [Project Grant Code: DP KPT FRGS/1/2021/SKK06/UM/02/4] and a University Research Grant [FF-2023-189] from UKM.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.