Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors.

It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

Specialized Intercellular Communications via Cytonemes and Nanotubes.

In recent years, thin membrane protrusions such as cytonemes and tunneling nanotubes have emerged as a novel mechanism of intercellular communication. Protrusion-based cellular interactions allow for specific communication between participating cells and have a distinct spectrum of advantages compared to secretion- and diffusion-based intercellular communication. Identification of protrusion-based signaling in diverse systems suggests that this mechanism is a ubiquitous and prevailing means of communication employed by many cell types. Moreover, accumulating evidence indicates that protrusion-based intercellular communication is often involved in pathogenesis, including cancers and infections. Here we review our current understanding of protrusion-based intercellular communication.

aristaless-like homeobox-3 is wound induced and promotes a low-Wnt environment required for planarian head regeneration.

The planarian Schmidtea mediterranea is a well-established model of adult regeneration, which is dependent on a large population of adult stem cells called neoblasts. Upon amputation, planarians undergo transcriptional wounding programs and coordinated stem cell proliferation to give rise to missing tissues. Interestingly, the Wnt signaling pathway is key to guiding what tissues are regenerated, yet less known are the transcriptional regulators that ensure proper activation and timing of signaling pathway components. Here, we have identified an aristaless-like homeobox transcription factor, alx-3, that is enriched in a population of putative neural-fated progenitor cells at homeostasis, and is also upregulated in stem cells and muscle cells at anterior-facing wounds upon amputation. Knockdown of alx-3 results in failure of head regeneration and patterning defects in amputated tail fragments. alx-3 is required for the expression of several early wound-induced genes, including the Wnt inhibitor notum, which is required to establish anterior polarity during regeneration. Together, these findings reveal a role for alx-3 as an early wound-response transcriptional regulator in both muscle cells and stem cells that is required for anterior regeneration by promoting a low-Wnt environment.

Modelling adult stem cells and their niche in health and disease with epithelial organoids.

Adult stem cells are responsible for homoeostasis and regeneration of epithelial tissues. Stem cell function is regulated by both cell autonomous mechanisms as well as the niche. Deregulated stem cell function contributes to diseases such as cancer. Epithelial organoid cultures generated from tissue-resident adult stem cells have allowed unprecedented insights into the biology of epithelial tissues. The subsequent adaptation of organoid technology enabled the modelling of the communication of stem cells with their cellular and non-cellular niche as well as diseases. Starting from its first model described in 2009, the murine small intestinal organoid, we discuss here how epithelial organoid cultures have been become a prime in vitro research tool for cell and developmental biology, bioengineering, and biomedicine in the last decade.

Division-Independent Differentiation of Muscle Stem Cells During a Growth Stimulus.

Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory.

Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Neuropeptide substance P alters stem cell fate to aid wound healing and promote epidermal stratification through asymmetric stem cell divisions.

Loss of sensory innervation delays wound healing and administration of the neuropeptide substance P improves re-epithelialization. Keratinocyte hyperproliferation post-wounding may result from symmetric stem cell (SC) self-renewal, asymmetric SC self-renewal, committed progenitor divisions, or a combination of these. However, the effects of sensory denervation and of neuropeptides on SC proliferation are not known. Here we show that early after wounding both asymmetric and symmetric SC self-renewal increase, without significant committed progenitor (CP) activation. Decreased sensory innervation is associated with a decrease in both SC and CP proliferation. Based on previous work showing that substance P is decreased in capsaicin-treated mice and improves wound healing in normal skin, we examined the effects of substance P on SC and CP proliferation during wound healing. Substance P restored asymmetric SC proliferation in skin with decreased sensory innervation, both at baseline and following wounding. Epidermis with decreased sensory innervation was severely thinned. Consistent with this, substance P-induced asymmetric SC proliferation resulted in increased stratification in skin with both normal and decreased innervation. Lapatinib prevented the substance P-induced increase in asymmetric SC divisions in murine epidermis, as well as the increase in epidermal stratification, suggesting that asymmetric SC divisions are required for epidermal stratification.

Cellular Heterogeneity in Adipose Tissues.

Adipose tissue depots in distinct anatomical locations mediate key aspects of metabolism, including energy storage, nutrient release, and thermogenesis. Although adipocytes make up more than 90% of adipose tissue volume, they represent less than 50% of its cellular content. Here, I review recent advances in genetic lineage tracing and transcriptomics that reveal the identities of the heterogeneous cell populations constituting mouse and human adipose tissues. In addition to mature adipocytes and their progenitors, these include endothelial and various immune cell types that together orchestrate adipose tissue development and functions. One salient finding is the identification of progenitor subtypes that can modulate adipogenic capacity through paracrine mechanisms. Another is the description of fate trajectories of monocyte/macrophages, which can respond maladaptively to nutritional and thermogenic stimuli, leading to metabolic disease. These studies have generated an extraordinary source of publicly available data that can be leveraged to explore commonalities and differences among experimental models, providing new insights into adipose tissues and their role in metabolic disease.

A role for partial epithelial-to-mesenchymal transition in enabling stemness in homeostasis and cancer.

Stem cells have self-renewal capacities and the ability to give rise to differentiated cells thereby sustaining tissues during homeostasis and injury. This structural hierarchy extends to tumours which harbor stem-like cells deemed cancer stem cells that propagate the tumour and drive metastasis and relapse. The process of epithelial-to-mesenchymal transition (EMT), which plays an important role in development and cancer cell migration, was shown to be correlated with stemness in both homeostasis and cancer indicating that stemness can be acquired and is not necessarily an intrinsic trait. Nowadays it is experimentally proven that the activation of an EMT program does not necessarily drive cells towards a fully mesenchymal phenotype but rather to hybrid E/M states. This review offers the latest advances in connecting the EMT status and stem-cell state of both non-transformed and cancer cells. Recent literature clearly shows that hybrid EMT states have a higher probability of acquiring stem cell traits. The position of a cell along the EMT-axis which coincides with a stem cell-like state is known as the stemness window. We show how the original EMT-state of a cell dictates the EMT/MET inducing programmes required to reach stemness. Lastly we present the mechanism of stemness regulation and the regulatory feedback loops which position cells at a certain EMT state along the EMT axis.

A transcriptionally repressed quiescence program is associated with paused RNA polymerase II and is poised for cell cycle re-entry.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition. This article has an associated First Person interview with the first authors of the paper.

Wnt/ß-catenin signalling is dispensable for adult neural stem cell homeostasis and activation.

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/ß-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here, we used Wnt/ß-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/ß-catenin signalling. Wnt/ß-catenin stimulation instructed the neuronal differentiation of proliferating NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, deletion of ß-catenin in NSCs did not affect either their activation or maintenance of their stem cell characteristics. Together, these results indicate that, although NSCs do respond to Wnt/ß-catenin stimulation in a dose-dependent and state-specific manner, Wnt/ß-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/ß-catenin signalling in adult hippocampal NSCs.

Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

How and Why the Circadian Clock Regulates Proliferation of Adult Epithelial Stem Cells.

First described in the early 20th century, diurnal oscillations in stem cell proliferation exist in multiple internal epithelia, including in the gastrointestinal tract, and in the epidermis. In the mouse epidermis, 3- to 4-fold more stem cells are in S-phase during the night than during the day. More recent work showed that an intact circadian clock intrinsic to keratinocytes is required for these oscillations in epidermal stem cell proliferation. The circadian clock also regulates DNA excision repair and DNA damage in epidermal stem cells in response to ultraviolet B radiation. During skin inflammation, epidermal stem cell proliferation is increased and diurnal oscillations are suspended. Here we discuss possible reasons for the evolution of this stem cell phenomenon. We argue that the circadian clock coordinates intermediary metabolism and the cell cycle in epidermal stem cells to minimize the accumulation of DNA damage from metabolism-generated reactive oxygen species. Circadian disruption, common in modern society, leads to asynchrony between metabolism and the cell cycle, and we speculate this will lead to oxidative DNA damage, dysfunction of epidermal stem cells, and skin aging.

Electroacupuncture Ameliorates Depression-Like Behaviors Comorbid to Chronic Neuropathic Pain via Tet1-Mediated Restoration of Adult Neurogenesis.

Although electroacupuncture (EA) stimulation is a widely used therapy for chronic pain and comorbid psychiatric disorders, its long-term effects on chronic neuropathic pain-induced depression and the underlying mechanisms remain elusive. In the present study, we found that EA stimulation was able to restore adult neurogenesis in the ventral dentate gyrus (DG), by both increasing neuronal differentiation and restoring the normal morphology of newborn dendrites, in mice with spared nerve injury surgery. By ablating the Nestin+ neural stem cells (NSCs) via diphtheria toxin fragment A expression, we further proved that neurogenesis in the ventral DG was crucial to the long-term, but not the immediate antidepressant effect of EA, nor was it associated with nociception. Furthermore, we found that the restoration of neurogenesis was dependent on Tet1-mediated epigenetic modification upon EA treatment. Tet1 could bind to the promoter of the Prox1 gene, thus catalyzing its demethylation and facilitating its expression, which finally contributed to the restoration of neurogenesis and amelioration of depression-like behaviors induced by chronic neuropathic pain. Thus, we conclude that EA stimulation restores inhibited Tet1 expression in hippocampal NSCs of mice with chronic neuropathic pain, and increased Tet1 expression ameliorates hypermethylation of Prox1 and restores normal adult neurogenesis in the ventral DG, which contributes to the long-term antidepressant effect of EA.

Identifying a Lung Stem Cell Subpopulation by Combining Single-Cell Morphometrics, Organoid Culture, and Transcriptomics.

Single-cell RNA sequencing is a valuable tool for dissecting cellular heterogeneity in complex systems. However, it is still challenging to estimate the proliferation and differentiation potentials of subpopulations within dormant tissue stem cells. Here, we established a new single-cell analysis method for profiling the organoid-forming capacity and differentiation potential of tissue stem cells to disclose stem cell subpopulations by integrating single-cell morphometrics, organoid-forming assay, and RNA sequencing, a method named scMORN. To explore lung epithelial stem cells, we initially developed feeder-free culture system, which could expand all major lung stem cells, including basal, club, and alveolar type 2 (AT2) cells, and found that club cells contained a subpopulation, which showed better survival rate and high proliferation capacity and could differentiate into alveolar cells. Using the scMORN method, we discovered a club cell subpopulation named Muc5b+ and large club (ML-club) cells that efficiently formed organoids than other club or AT2 cells in our feeder-free organoid culture and differentiated into alveolar cells in vitro. Single-cell transcriptome profiling and immunohistochemical analysis revealed that ML-club cells localized at the intrapulmonary proximal airway and distinct from known subpopulations of club cells such as BASCs. Furthermore, we identified CD14 as a cell surface antigen of ML-club cells and showed that purified CD14+ club cells engrafted into injured mouse lungs had better engraftment rate and expansion than other major lung stem cells, reflecting the observations in organoid culture systems. The scMORN method could be adapted to different stem cell tissues to discover useful stem-cell subpopulations.

Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration - A review.

Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.

Single-cell expression profile of Drosophila ovarian follicle stem cells illuminates spatial differentiation in the germarium.

BACKGROUND: How stem cell populations are organized and regulated within adult tissues is important for understanding cancer origins and for developing cell replacement strategies. Paradigms such as mammalian gut stem cells and Drosophila ovarian follicle stem cells (FSC) are characterized by population asymmetry, in which stem cell division and differentiation are separately regulated processes. These stem cells behave stochastically regarding their contributions to derivative cells and also exhibit dynamic spatial heterogeneity. Drosophila FSCs provide an excellent model for understanding how a community of active stem cells maintained by population asymmetry is regulated. Here, we use single-cell RNA sequencing to profile the gene expression patterns of FSCs and their immediate derivatives to investigate heterogeneity within the stem cell population and changes associated with differentiation. RESULTS: We describe single-cell RNA sequencing studies of a pre-sorted population of cells that include FSCs and the neighboring cell types, escort cells (ECs) and follicle cells (FCs), which they support. Cell-type assignment relies on anterior-posterior (AP) location within the germarium. We clarify the previously determined location of FSCs and use spatially targeted lineage studies as further confirmation. The scRNA profiles among four clusters are consistent with an AP progression from anterior ECs through posterior ECs and then FSCs, to early FCs. The relative proportion of EC and FSC clusters are in good agreement with the prevalence of those cell types in a germarium. Several genes with graded profiles from ECs to FCs are highlighted as candidate effectors of the inverse gradients of the two principal signaling pathways, Wnt and JAK-STAT, that guide FSC differentiation and division. CONCLUSIONS: Our data establishes an important resource of scRNA-seq profiles for FSCs and their immediate derivatives that is based on precise spatial location and functionally established stem cell identity, and facilitates future genetic investigation of regulatory interactions guiding FSC behavior.

Efficacy of injection of autologous adipose tissue in the treatment of patients with complex and recurrent fistula-in-ano of cryptoglandular origin.

BACKGROUND: Adipose tissue injections, a rich source of mesenchymal stem cells, have been successfully used to promote anal fistula healing. This study aimed to investigate the efficacy of adipose tissue injection in treating patients with complex and recurrent fistulas of cryptoglandular origin. METHODS: We conducted a prospective, single-center, open-label, non-randomized, interventional clinical trial from January 2020 to December 2022. We enrolled nine patients, who were evaluated after at least 12 months of follow-up. All patients had seton removal, fistula tract excision or curettage, and a mucosal flap if possible or, alternatively, an internal opening suture. We used a commercially available system to collect and process adipose tissue prior to injection. This system allowed the collection, microfragmentation, and filtration of tissue. RESULTS: Selected cases included six men and three women with a median age of 42 (range 31-55) years. All patients had an extended disease course period, ranging from 3 to 13 (mean 6.6) years, and a history of multiple previous surgeries, including two to eight interventions (a mean of 4.4 per case). All fistulas were high transsphincteric, four cases horseshoe and two cases with secondary suprasphincteric or peri-elevator tract fistulas. Six cases (66%) achieved complete fistula healing at a mean follow-up of 18 (range 12-36) months. Three cases (33.3%) experienced reduced secretion and decreased anal discomfort. CONCLUSIONS: In patients with complex and recurrent fistulas, such as the ones described, many from palliative treatments with setons, the adjuvant injection of adipose tissue might help achieve complete healing or improvement in a significant percentage of cases. CLINICALTRIALS: The study protocol was prospectively registered on ClinicalTrials.gov (NCT04750499).

Differential Colonization and Mucus Ultrastructure Visualization in Bovine Ileal and Rectal Organoid-Derived Monolayers Exposed to Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.