Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

BackgroundWe previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.MethodsHuman small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.ResultsCollagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.ConclusionType I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

Background Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. While techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Methods Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM), and glycogen synthase kinase 3 inhibitor (GSK3i) and studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry (IHC) and quantitative PCR (qPCR). Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Results Whereas ENR medium supported murine enteroid growth; it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Discussion We describe a method to maintain juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced using lentiviral vectors.

IntroductionMedical students are the source of a country's physicians. Determining how medical students select their areas of specialization is the key to achieve a balanced distribution of doctors among all specialties. The objective is to identify the number of medical students who have decided their postgraduate specialty career, their career specialties preference, and factors that may influence their decision to select a particular specialty.MethodsA facility based cross-sectional study was conducted in September 2013 at Faculty of Medicine, University of Medical Sciences and Technology, Khartoum, Sudan. A self-administered semi-structured questionnaire comprising demographic data and questions about future specialties preferences and factors influencing those preferences was distributed to 887 male and female students, (from first to fifth academic years) recruited in the study.ResultsResponse rate was 73% with 647 questionnaires collected, out of 887 eligible medical students. Of the returned questionnaires, 604 were valid. The majority of students (541, 89.6%) have chosen a specialty. Surgery, medicine, paediatrics and obstetrics and gynecology were the most selected specialties. The least selected specialty was anaesthesiology. A significant association was found between gender and specialty choice using Chi-square test (p = 0.00). There was no association between undergraduate level and specialty choice (p = 0.633). The most common reason for choosing a specific specialty was “Personal Interest” (215, 39.7%) followed by being “Helpful to the community” (144, 26.6%).ConclusionSurgery, medicine, paediatrics and obstetrics and gynecology were the most selected specialties.

Multilevel-thresholding is an efficient method used in image segmentation. This paper presents a hybrid meta-heuristic approach for multi-level thresholding image segmentation by integrating both the artificial bee colony (ABC) algorithm and the sine-cosine algorithm (SCA). The proposed algorithm, called ABCSCA, is applied to segment images and it utilizes Otsu's function as the objective function. The proposed ABCSCA uses ABC to optimize the threshold and to reduce the search region. Thereafter, the SCA algorithm uses the output of ABC to determine the global optimal solution, which represents the thresholding values. To evaluate the performance of the proposed ABCSCA, a set of experimental series is performed using nineteen images. In the first experimental series, the proposed ABCSCA is assessed at the low threshold levels and compared with the ABC and SCA as traditional methods. Moreover, the second experimental series aims to evaluate the ABCSCA at high threshold levels and it is compared with six algorithms in addition to the SCA and ABC. Besides, the proposed method is evaluated using the fuzzy entropy. The results demonstrate the effectiveness of the proposed algorithm and showed that it outperforms other algorithms in terms of performance measures, such as Peak Signal-to-Noise Ratio (PSNR) and the Structural Similarity Index (SSIM).INDEX TERMS Image segmentation, multi-level thresholding, artificial bee colony (ABC), sine-cosine algorithm (SCA).