Nicholas R. Smith

Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

BACKGROUND & AIMS Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

Correlation of the same fields imaged in the TEM, confocal, LM, and microCT by image registration: from specimen preparation to displaying a final composite image.

Correlated imaging is the process of imaging a specimen with two complementary modalities and then registering and overlaying the fields obtained in each modality to create a composite view. One of the images is made somewhat transparent, allowing detail in the underlying image to be visible and assisting in the registration of the two images. As an example, an image localizing a specific tissue component by fluorescence may be overlaid atop a TEM image of the same field. The resulting composite image would demonstrate specific ultrastructural features in the high-resolution TEM field, which are colorized in the overlay. Other examples include composites from MicroCT or soft X-ray images overlaid atop light microscopy or TEM images. Automated image registration may be facilitated by a variety of sophisticated computer programs utilized by high-throughput laboratories. This chapter is meant for the more occasional user wishing to align images manually. ImageJ is a public domain, image processing program developed at the National Institutes of Health and is available to anyone as a free download. ImageJ performs marvelously well for the purpose of image registration; therefore, step-by-step instructions are included here. Specimen handling, including fixation and choice of embedding media, is not straightforward for correlative imaging. A step-by-step description of the protocols which work in our laboratory is included for simultaneous localization in LM, EM and micro-CT, as well as maintaining GFP emission in tissue embedded for TEM.

Defining a stem cell hierarchy in the intestine: markers, caveats and controversies

The past decade has appreciated rapid advance in identifying the once elusive intestinal stem cell (ISC) populations that fuel the continual renewal of the epithelial layer. This advance was largely driven by identification of novel stem cell marker genes, revealing the existence of quiescent, slowly‐ and active‐cycling ISC populations. However, a critical barrier for translating this knowledge to human health and disease remains elucidating the functional interplay between diverse stem cell populations. Currently, the precise hierarchical and regulatory relationships between these ISC populations are under intense scrutiny. The classical theory of a linear hierarchy, where quiescent and slowly‐cycling stem cells self‐renew but replenish an active‐cycling population, is well established in other rapidly renewing tissues such as the haematopoietic system. Efforts to definitively establish a similar stem cell hierarchy within the intestinal epithelium have yielded conflicting results, been difficult to interpret, and suggest non‐conventional alternatives to a linear hierarchy. While these new and potentially paradigm‐shifting discoveries are intriguing, the field will require development of a number of critical tools, including highly specific stem cell marker genes along with more rigorous experimental methodologies, to delineate the complex cellular relationships within this dynamic organ system.

Epithelial and mesenchymal contribution to the niche: a safeguard for intestinal stem cell homeostasis

The intestinal stem cell (ISC) niche is a highly complex microenvironment that supports stem cell maintenance and precisely orchestrates the balance between proliferation and differentiation. Here, multiple signaling molecules emanating from both the underlying mesenchyme and crypt-based epithelial cells cooperate to govern stem cell homeostasis (Figure 1). Understanding the cellular and molecular constituents of the niche is of critical importance to harness stem cells for tissue regeneration and to elucidate disease mechanisms. The conserved Wnt, Notch and epidermal growth factor (EGF) signaling pathways are identified regulators of stem cell behavior, however resolution of the cellular players and the impact of signaling molecules on different progenitor populations residing within the niche remain poorly understood. In this issue of Gastroenterology, a group led by Hans Clevers (Farin et al.)[1] specifically address the niche requirement of Paneth cell (PC)-derived Wnt3 in mediating intestinal homeostasis. Analysis of Wnt3-null epithelia in both in vivo, and in vitro cultures resulted in no apparent effect within the intact small intestine, but lack of growth in the culture system. While PCs have been defined to express Wnt6 and 9b in vivo, PCs did not express adequate levels to rescue the phenotype in vitro; instead, addition of the mesenchymally-expressed Wnt2b ligand supported enteroid growth. These findings highlight the robust compensatory signaling support provided by the underlying mesenchymal niche cells despite loss of an epithelial Wnt source. Figure 1 Comparison of in vivo (left) and in vitro (right) signaling support to the intestinal stem cell. Signaling networks between the Paneth cell and stem cell appear to be broadly conserved. However, disruption on one element of a single pathway highlights ...

Cell Adhesion Molecule CD166/ALCAM Functions Within the Crypt to Orchestrate Murine Intestinal Stem Cell Homeostasis

Background & Aims Intestinal epithelial homeostasis is maintained by active-cycling and slow-cycling stem cells confined within an instructive crypt-based niche. Exquisite regulating of these stem cell populations along the proliferation-to-differentiation axis maintains a homeostatic balance to prevent hyperproliferation and cancer. Although recent studies focus on how secreted ligands from mesenchymal and epithelial populations regulate intestinal stem cells (ISCs), it remains unclear what role cell adhesion plays in shaping the regulatory niche. Previously we have shown that the cell adhesion molecule and cancer stem cell marker, CD166/ALCAM (activated leukocyte cell adhesion molecule), is highly expressed by both active-cycling Lgr5+ ISCs and adjacent Paneth cells within the crypt base, supporting the hypothesis that CD166 functions to mediate ISC maintenance and signal coordination. Methods Here we tested this hypothesis by analyzing a CD166–/– mouse combined with immunohistochemical, flow cytometry, gene expression, and enteroid culture. Results We found that animals lacking CD166 expression harbored fewer active-cycling Lgr5+ ISCs. Homeostasis was maintained by expansion of the transit-amplifying compartment and not by slow-cycling Bmi1+ ISC stimulation. Loss of active-cycling ISCs was coupled with deregulated Paneth cell homeostasis, manifested as increased numbers of immature Paneth progenitors due to decreased terminal differentiation, linked to defective Wnt signaling. CD166–/– Paneth cells expressed reduced Wnt3 ligand expression and depleted nuclear β-catenin. Conclusions These data support a function for CD166 as an important cell adhesion molecule that shapes the signaling microenvironment by mediating ISC–niche cell interactions. Furthermore, loss of CD166 expression results in decreased ISC and Paneth cell homeostasis and an altered Wnt microenvironment.

Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell Populations

Background & Aims Continual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein–coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function. Methods We used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC–green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed. Results Two novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established. Conclusions These data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

Building Blocks for Engineering the Small Intestine

Bowel transplantation represents the current standard of care for patients suffering from severe intestinal disorders such as short bowel syndrome. Despite improvements in surgical procedures, there remains an approximate 50% 5-year survival rate for intestinal transplant patients [55]. Currently, the primary barriers to successful organ transplantation are the lack of transplantable tissue, the potential for transplant rejection, and the requirement of lifelong immunosuppression [35]. These challenges highlight the need to fully explore the developing field of intestinal epithelial stem cell biology and their ability for ex vivo expansion of tissue in order to reach the ultimate goal of engineering personalized transplantable intestine from patient-derived cells.