Deriving functional human enteroendocrine cells from pluripotent stem cells.

Enteroendocrine cells (EECs) are a minor cell population in the intestine yet they play a major role in digestion, satiety and nutrient homeostasis. Recently developed human intestinal organoid models include EECs, but their rarity makes it difficult to study their formation and function. Here, we used the EEC-inducing property of the transcription factor NEUROG3 in human pluripotent stem cell-derived human intestinal organoids and colonic organoids to promote EEC development in vitro An 8-h pulse of NEUROG3 expression induced expression of known target transcription factors and after 7 days organoids contained up to 25% EECs in the epithelium. EECs expressed a broad array of human hormones at the mRNA and/or protein level, including motilin, somatostatin, neurotensin, secretin, substance P, serotonin, vasoactive intestinal peptide, oxyntomodulin, GLP-1 and INSL5. EECs secreted several hormones including gastric inhibitory polypeptide (GIP), ghrelin, GLP-1 and oxyntomodulin. Injection of glucose into the lumen of organoids caused an increase in both GIP secretion and K-cell number. Lastly, we observed formation of all known small intestinal EEC subtypes following transplantation and growth of human intestinal organoids in mice.

Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells.

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.

Implementation and Evaluation of Adverse Childhood Experiences Screening in Pediatrics and Obstetrics Settings.

BACKGROUND: Screening for adverse childhood experiences (ACEs) in prenatal and pediatric populations is recommended by the California ACEs Aware initiative and is a promising practice to interrupt ACEs in children and mitigate ACEs-related health complications in children and families. Yet, integrating ACEs screening into clinical practice poses several challenges. OBJECTIVE: The objective of this report was to evaluate the Kaiser Permanente Northern California and Kaiser Permanente Southern California pilots and implementation of ACEs screening into routine prenatal (Kaiser Permanente Northern California) and pediatric (Kaiser Permanente Southern California) care. MATERIALS AND METHODS: These pilots were evaluated and compared to identify common challenges to implementation and offer promising practices for negotiating these challenges. Evaluation methods included feedback from staff, clinicians, and patients, as well as comparisons of methods to overcome various barriers to screening implementation. RESULTS: Implementing ACEs screening, like implementation of any new component of clinical care, takes careful planning, education, creation of content and workflows, and continuous integration of feedback from both patients and staff. CONCLUSION: This evaluation can serve as support for care teams who are considering implementing ACEs screening or who are already screening for ACEs. More research is needed regarding the relationship between ACEs and preventable and treatable health outcomes to improve health for patients and their families.

Association of Adverse Childhood Experiences and Resilience With Depression and Anxiety During Pregnancy.

This was a retrospective cohort study of pregnant individuals in the Kaiser Permanente Northern California system who were screened for adverse childhood experiences and resilience as part of standard prenatal care at about 16 weeks of gestation. Overall, 14,625 pregnancies were included; 17.0% had newly identified depression; 9.8% had newly identified depression symptoms; and 8.9% had newly identified anxiety during the pregnancy with no known preexisting diagnosis. We found that adverse childhood experiences and low resilience were independently associated with newly identified depressive disorders, depression symptoms, and anxiety disorders during pregnancy. When adverse childhood experiences and resilience were modeled in combination, the greatest odds of each outcome occurred in individuals with a combination of four or more adverse childhood experiences and low resilience (vs no adverse childhood experiences and high resilience): depression adjusted odds ratio (aOR) 6.43 (95% CI, 5.23-7.90), depression symptoms aOR 9.49 (95% CI, 7.50-12.0), and anxiety disorder aOR 4.79 (95% CI, 3.81-6.02). Routine screening for adverse childhood experiences and resilience may identify individuals at risk of developing prenatal depression and anxiety, allowing faster resource linkage and potentially improved maternal and child outcomes.

Association of the COVID-19 Pandemic With Unstable and/or Unsafe Living Situations and Intimate Partner Violence Among Pregnant Individuals.

Importance: The social, behavioral, and economic consequences of the COVID-19 pandemic may be associated with unstable and/or unsafe living situations and intimate partner violence (IPV) among pregnant individuals. Objective: To investigate trends in unstable and/or unsafe living situations and IPV among pregnant individuals prior to and during the COVID-19 pandemic. Design, Setting, and Participants: A cross-sectional population-based interrupted time-series analysis was conducted among Kaiser Permanente Northern California members who were pregnant and screened for unstable and/or unsafe living situation and IPV as part of standard prenatal care between January 1, 2019, and December 31, 2020. Exposures: COVID-19 pandemic (prepandemic period: January 1, 2019, to March 31, 2020; during pandemic period: April 1 to December 31, 2020). Main Outcomes and Measures: The 2 outcomes were unstable and/or unsafe living situations and IPV. Data were extracted from electronic health records. Interrupted time-series models were fit and adjusted for age and race and ethnicity. Results: The study sample included 77 310 pregnancies (74 663 individuals); 27.4% of the individuals were Asian or Pacific Islander, 6.5% were Black, 29.0% were Hispanic, 32.3% were non-Hispanic White, and 4.8% were other/unknown/multiracial, with a mean (SD) age of 30.9 (5.3) years. Across the 24-month study period there was an increasing trend in the standardized rate of unsafe and/or unstable living situations (2.2%; rate ratio [RR], 1.022; 95% CI, 1.016-1.029 per month) and IPV (4.9%; RR, 1.049; 95% CI, 1.021-1.078 per month). The ITS model indicated a 38% increase (RR, 1.38; 95% CI, 1.13-1.69) in the first month of the pandemic for unsafe and/or unstable living situation, with a return to the overall trend afterward for the study period. For IPV, the interrupted time-series model suggested an increase of 101% (RR, 2.01; 95% CI, 1.20-3.37) in the first 2 months of the pandemic. Conclusions and Relevance: This cross-sectional study noted an overall increase in unstable and/or unsafe living situations and IPV over the 24-month period, with a temporary increase associated with the COVID-19 pandemic. It may be useful for emergency response plans to include IPV safeguards for future pandemics. These findings suggest the need for prenatal screening for unsafe and/or unstable living situations and IPV coupled with referral to appropriate support services and preventive interventions.

Associations between Adverse Childhood Experiences (ACEs) and Prenatal Mental Health and Substance Use.

Adverse childhood experiences (ACEs) are common and increase the risk of poor health outcomes. Resilience may offer protection against the impacts of ACEs. This study examined the association between maternal ACEs and mental/behavioral health outcomes during pregnancy overall and by resilience. The sample comprised pregnant patients in two pilot studies screened for eight ACEs and resilience during standard prenatal care in Kaiser Permanente Northern California from 1 March 2016 to 30 July 2016 (Study 1, medical centers A, B) and from 1 April 2018 to 31 March 2019 (Study 2, medical centers A, C). Early pregnancy outcomes included anxiety and depressive disorders, depression symptoms, intimate partner violence (IPV), and substance use. Multivariable logistic regression was used in this cross-sectional study to examine associations between maternal ACEs (0, 1-2, ≥3) and mental/behavioral health outcomes overall and among those with low and high resilience. Patients (n = 1084) averaged 30.8 years (SD 5.1); 41.7% were non-Hispanic White; 41.7% experienced ≥1 ACE, and 40.3% had low resilience. Patients with 1-2 ACEs or ≥3 ACEs (versus 0 ACEs) had higher odds of anxiety and depressive disorders, depressive symptoms, IPV, and any prenatal substance use (OR 1.44-4.40, p < 0.05). Each individual ACE was associated with ≥2 mental/behavioral health outcomes. In stratified analyses, having ≥1 ACE (vs. 0) was associated with a greater number of mental/behavioral health outcomes among patients with low versus high resilience. ACEs were associated with prenatal mental/behavioral health conditions, particularly in the context of low resilience, highlighting the importance of trauma-informed prenatal care and the need to study resilience-building interventions during pregnancy.

Pregnant women's perspectives on screening for adverse childhood experiences and resilience during prenatal care.

OBJECTIVE: To examine the acceptability of routine screening for adverse childhood experiences (ACEs) and resilience during prenatal care. METHOD: This study examined pregnant women's perspectives (N = 119) on ACEs and resilience screening during prenatal care in two medical centers via postscreening telephone surveys. Chi-square tests and Fisher's Exact Tests examined whether responses varied with ACEs (0 [62.2%], 1-2 [21.0%], 3+ [16.8%]) or resilience (high [64.7%] vs. low [35.3%]). RESULTS: The sample (N = 119) was 36.1% non-Hispanic White, 26.1% Hispanic, 8.4% Black, 23.5% Asian/Pacific Islander, and 5.9% Other, with a median age of 31 (IQR: 28-34) and average neighborhood median household income of $100,734 (SD = $37,079). Most women thought prenatal care should include conversations about ACEs (82.2%) and resilience (94.0%) and very strongly believed that good coping skills can help reduce the harmful effects of childhood stress (79.0%). Nearly half (41.2%) used ≥1 mental health resource from the resource handout provided at screening. Some women thought conversations could be improved if they took place with a mental health professional (37.3%), with more provider empathy (40.7%), more education about ACEs and health (55.1%), and if the screening included additional stressors (53.4%). Notably, most women (73.5%) would like their partner to also receive the screening. Women with more ACEs were more likely to want a longer conversation, and those with low versus high resilience were more likely to prefer that a mental health professional conduct the screening. CONCLUSIONS: Results indicate that women value ACEs screening during prenatal care and provide actionable recommendations to improve future screenings and discussions. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Adverse Childhood Experiences and Pregnancy Intentions among Pregnant Women Seeking Prenatal Care.

BACKGROUND: This study examined whether adverse childhood experiences (ACEs) are associated with increased risk of having an unwanted or mistimed pregnancy. METHODS: Women in two medical centers within an integrated health system were screened for ACEs during standard prenatal care (N = 745). Multinomial multivariable logistic regression analyses examined the associations of ACEs (count and type) with pregnancy intentions, adjusting for covariates. RESULTS: Overall, 58.3% of pregnant women reported no ACEs, 19.1% reported one ACE, and 22.7% reported two or more ACEs; 76.2% reported wanting to get pregnant, 18.5% reported wanting to get pregnant but not at this time (i.e., mistimed pregnancy), and 5.2% reported not wanting to get pregnant at all (i.e., unwanted pregnancy). Having two or more (vs. 0) ACEs was associated with higher odds of an unwanted pregnancy (odds ratio, 2.60; 95% confidence interval, 1.19-5.68). Further, childhood loss of parent (odds ratio, 2.20; 95% confidence interval, 1.03-4.71) and neglect (odds ratio, 5.67; 95% confidence interval, 1.72-18.72) were each associated with higher odds of an unwanted pregnancy in separate analyses. ACEs count and type were not significantly associated with having a mistimed pregnancy. CONCLUSIONS: Among women screened for ACEs during standard prenatal care, ACEs were associated with increased odds of having an unwanted pregnancy, but not a mistimed pregnancy. Additional research is needed to better understand the mechanisms through which ACEs and other individual, social, and contextual factors impact pregnancy intentions to better support women and provide appropriate resources to help prevent unintended pregnancies.

Adverse Childhood Experiences and Early and Continued Breastfeeding: Findings from an Integrated Health Care Delivery System.

Purpose: To examine whether adverse childhood experiences (ACEs) are associated with breastfeeding behaviors. Methods: Women in three Kaiser Permanente Northern California medical centers were screened for ACEs during standard prenatal care (N = 926). Multivariable binary and multinomial logistic regression was used to test whether ACEs (count and type) were associated with early breastfeeding at the 2-week newborn pediatric visit and continued breastfeeding at the 2-month pediatric visit, adjusting for covariates. Results: Overall, 58.2% of women reported 0 ACEs, 19.2% reported 1 ACE, and 22.6% reported 2+ ACEs. Two weeks postpartum, 92.2% reported any breastfeeding (62.9% exclusive, 29.4% mixed breastfeeding/formula). Compared with women with 0 ACEs, those with 2+ ACEs had increased odds of any breastfeeding (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 1.3-5.6) and exclusive breastfeeding 2 weeks postpartum (OR = 3.0, 95% CI = 1.4-6.3). Among those who breastfed 2 weeks postpartum, 86.4% reported continued breastfeeding (57.5% exclusive, 28.9% mixed breastfeeding/formula) 2 months postpartum. ACE count was not associated with continued breastfeeding 2 months postpartum. Individual ACEs were not related to breastfeeding outcomes, with the exception that living with someone who went to jail or prison was associated with lower odds of continued breastfeeding 2 months postpartum. Conclusions: ACE count was associated with greater early breastfeeding, but not continued breastfeeding, among women screened for ACEs as part of standard prenatal care. Results reiterate the need to educate and assist all women to meet their breastfeeding goals, regardless of ACE score.

Adverse Childhood Experiences and Mental and Behavioral Health Conditions During Pregnancy: The Role of Resilience.

INTRODUCTION: Little is known about how exposure to adverse childhood experiences (ACEs) and protective factors, such as resilience, influence prenatal mental and behavioral health. This study examined associations between exposure to ACEs and mental and behavioral health during pregnancy overall and among women with high versus low levels of resilience. MATERIALS AND METHODS: Women in two Kaiser Permanente Northern California medical centers were screened for ACEs and resilience during prenatal care (∼14-23 weeks of gestation; N = 355). Multivariable logistic regression analyses examined associations between ACEs and prenatal mental and behavioral health conditions overall and for women with low (≤32) versus high (>32) resilience on the 10-item Connor-Davidson Resilience Scale. RESULTS: Overall, 54% of women reported 0 ACEs, 28% 1-2 ACEs, and 18% 3+ ACEs. Relative to women with 0 ACEs, those with 1-2 ACEs had higher odds of an anxiety or depressive disorder and intimate partner violence (IPV) (odds ratios [ORs] 2.42-3.12, p < 0.05), and those with 3+ ACEs had higher odds of an anxiety or depressive disorder, depression symptoms, and IPV (ORs 3.08-4.71, p < 0.05). In stratified analyses by high (56%) and low (44%) resilience, having one or more ACEs (vs. 0 ACEs) was only associated with worse mental and behavioral health in women with low resilience. CONCLUSIONS: ACEs predicted mental and behavioral health conditions among pregnant women, and associations were the strongest among women with low levels of current resilience. Longitudinal research is needed to understand the causal mechanisms underlying these associations.

Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis.

Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5-CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/ß-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.

Feasibility and Acceptability of Screening for Adverse Childhood Experiences in Prenatal Care.

INTRODUCTION: Adverse childhood experiences (ACEs) are common among pregnant women and contribute to increased risk for negative perinatal outcomes, yet few clinicians screen prenatal patients for ACEs. The purpose of this study was to evaluate the feasibility and acceptability of screening for ACEs in standard prenatal care. METHODS: We evaluated a 4-month pilot (March 2016-June 2016) to screen pregnant women (at ∼14-23 weeks of gestation) for ACEs and resiliency in two Kaiser Permanente Northern California medical centers (N = 480). We examined the acceptability of the screening to patients through telephone surveys (N = 210) and to clinicians through surveys and focus groups (N = 26). RESULTS: Most eligible patients (78%) were screened. Patients who received the screening were significantly more likely to be non-Hispanic White, Asian, or of "Other" or "Unknown" race/ethnicity than African American or Hispanic race/ethnicity (p = 0.02). Among those screened, 88% completed the questionnaires; 54% reported 0 ACEs, 28% reported 1-2 ACEs, and 18% reported ≥3 ACEs. Most patients were somewhat or very comfortable completing the questionnaires (91%) and discussing ACEs with their clinician (93%), and strongly or somewhat strongly agreed that clinicians should ask their prenatal patients about ACEs (85%). Clinicians reported significant pre- to postpilot increases in comfort discussing ACEs, providing education, and offering resources (ps < 0.01). Clinicians' willingness to screen for ACEs was contingent on adequate training, streamlined workflows, inclusion of resilience screening, and availability of mental health, parenting, and social work resources. CONCLUSION: ACEs screening as part of standard prenatal care is feasible and generally acceptable to patients. Women's health clinicians are willing to screen patients for ACEs when appropriately trained and adequate behavioral health referral resources are available.

Differentiation of Human Pluripotent Stem Cells into Colonic Organoids via Transient Activation of BMP Signaling.

Gastric and small intestinal organoids differentiated from human pluripotent stem cells (hPSCs) have revolutionized the study of gastrointestinal development and disease. Distal gut tissues such as cecum and colon, however, have proved considerably more challenging to derive in vitro. Here we report the differentiation of human colonic organoids (HCOs) from hPSCs. We found that BMP signaling is required to establish a posterior SATB2+ domain in developing and postnatal intestinal epithelium. Brief activation of BMP signaling is sufficient to activate a posterior HOX code and direct hPSC-derived gut tube cultures into HCOs. In vitro, HCOs express colonic markers and contained colon-specific cell populations. Following transplantation into mice, HCOs undergo morphogenesis and maturation to form tissue that exhibits molecular, cellular, and morphologic properties of human colon. Together these data show BMP-dependent patterning of human hindgut into HCOs, which will be valuable for studying diseases including colitis and colon cancer.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

In vivo Fluorescein Isothiocyanate-dextran (FD4) Permeability Assay.

Using pluripotent stem cells, it is now becoming possible to develop tissue models of organ systems within the body. These organs will allow for the study of organ function, physiology, embryology, and even pathologic processes. Recently, our group developed a model of human small intestine developed from human pluripotent stem cells which when transplanted in vivo, produce a mature, cystic intestinal structure that has digestive functions similar to that of native small intestine (Watson et al., 2014). Intestinal permeability is a primordial function of both the epithelium and associated tight junctions to control nutrient intake and prevent the passage of pathogens. One way to study gastrointestinal paracellular permeability is by determining the ability of fluorophores-conjugated macromolecules (i.e., fluorescein isothiocyanate-dextran (FITC-dextran; or FD4) to cross from the lumen and into circulation (Dong et al., 2014). We were able to test the intestinal permeability by injecting FITC-dextran directly into the lumen of the bioengineered intestine and determining the fluorescence within the blood of the murine host at various time points after injection.

Intravenous Tomato Lectin Injection to Assess Functional Vasculature.

Pluripotent stem cells have recently allowed for the development of tissue models for the various organ systems within the body. These models allow scientists to study organ function, physiology, embryology, and even pathologic processes. Studies on tissue can be done in vitro and/or transplanted into animal models for studies in vivo. Recently, our lab developed a model of human small intestine derived from human pluripotent stem cells which when transplanted in vivo, matured into an intestinal structure similar to that of adult intestine. The maturity of the transplanted human intestinal tissue was dependent upon the development of an adequate blood supply primarily from the murine host. In order to better study the developed vascular network within our transplanted intestinal tissue, we injected Fluorescein labeled Lycopersicon esculentum (tomato) lectin into the mouse tail vein (Watson et al., 2014). Using the property of this lectin to bind to the endothelium, we were able to visualize the vasculature within the transplant.

Chemokine Involvement in Fetal and Adult Wound Healing.

Significance: Fetal wounds heal with a regenerative phenotype that is indistinguishable from surrounding skin with restored skin integrity. Compared to this benchmark, all postnatal wound healing is impaired and characterized by scar formation. The biologic basis of the fetal regenerative phenotype can serve as a roadmap to recapitulating regenerative repair in adult wounds. Reduced leukocyte infiltration, likely mediated, in part, through changes in the chemokine milieu, is a fundamental feature of fetal wound healing. Recent Advances: The contributions of chemokines to wound healing are a topic of active investigation. Recent discoveries have opened the possibility of targeting chemokines therapeutically to treat disease processes and improve healing capability, including the possibility of achieving a scarless phenotype in postnatal wounds. Critical Issues: Successful wound healing is a complex process, in which there is a significant interplay between multiple cell types, signaling molecules, growth factors, and extracellular matrix. Chemokines play a crucial role in this interplay and have been shown to have different effects in various stages of the healing process. Understanding how these chemokines are locally produced and regulated during wound healing and how the chemokine milieu differs in fetal versus postnatal wounds may help us identify ways in which we can target chemokine pathways. Future Directions: Further studies on the role of chemokines and their role in the healing process will greatly advance the potential for using these molecules as therapeutic targets.

Establishment of human epithelial enteroids and colonoids from whole tissue and biopsy.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed "enteroids" when derived from small intestine and "colonoids" when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The Basic Helix-Loop-Helix Transcription Factor NEUROG3 Is Required for Development of the Human Endocrine Pancreas.

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast, humans with NEUROG3 mutations are born with endocrine pancreas function, calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly, we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover, NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast, a 75-90% knockdown of NEUROG3 caused a reduction, but not a loss, of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells.