Nambirajan Sundaram

An in vivo model of human small intestine using pluripotent stem cells

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

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 Hirschsprungs 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.

High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

Intestinal crypts reproducibly expand in culture.

BACKGROUND In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro. METHODS AND MATERIALS Crypts were harvested from 5 cm of proximal, middle, and distal small intestine of C57BL/6J mice aged 4 wk, 6-8 wk, 12-14 wk, and 18-20 wk (n = 4-6 animals) and cultured. For each region, we determined the efficiency of crypts forming enterospheres (day 1) and progressing to enteroids (day 7). Subsequently, enteroids were passaged and cryopreserved to determine if growth was changed by these manipulations. RESULTS Forty-three to 99% of intestinal crypts formed enterospheres, with higher efficiency in proximal small intestine and in younger mice. Twenty-five to 64% of enterospheres progressed to budding enteroids within 7 d. In vitro expansion was greater in proximal enteroids. This expansion continued in a logarithmic fashion, with ≈ 97% replating efficiency of isolated enteroid crypt buds. Following cryopreservation, ≈ 90% of enteroids recovered normal proliferative capacity. CONCLUSIONS Intestinal crypt culture is efficient and significantly expands intestinal tissue in a reproducible manner. Regional and age growth differences may reflect distinct stem cell characteristics or differences in support cells. The ability to culture and expand intestinal tissue in vitro provides a potential translational approach toward understanding and treating patients with short bowel syndrome.

Biomarkers for early detection of sickle nephropathy

Renal complications affect nearly 30–50% of adults with sickle cell anemia (SCA), causing significant morbidity and mortality. Standard renal function tests like serum creatinine and glomerular filtration rate become abnormal in this disease only when renal damage has become extensive and largely irreversible. Moreover, not all patients develop sickle nephropathy (SN). Therefore, noninvasive biomarkers that predict early onset of SN are necessary. We performed a cross‐sectional analysis for nephropathy in 116 patients with sickle cell disease, analyzing urinary kidney injury molecule‐1 (KIM‐1), liver‐type fatty acid binding protein (L‐FABP), N‐acetyl‐b‐D‐glucosaminidase (NAG), neutrophil gelatinase‐associated lipocalin (NGAL) and transforming growth factor‐β1 (TGF‐β), together with conventional renal biomarkers (urine albumin and osmolality, and serum creatinine and cystatin C estimated GFR) during routine clinic visits when patients were at steady‐state/baseline. We observed a distinct biomarker pattern: KIM‐1 and NAG emerged as biomarkers with a strong association with albuminuria. Surprisingly, and in contrast to other acute/chronic renal disorders, NGAL, L‐FABP, and TGF‐β levels did not show any relationship with albuminuria in patients with SCA. Our study identifies potential biomarkers for SN, and suggests longitudinal validation of these biomarkers for early detection of SN, so that therapeutic interventions can be applied before renal damage becomes irreversible. Am. J. Hematol. 2011.

Placenta growth factor (PlGF), a novel inducer of plasminogen activator inhibitor-1 (PAI-1) in sickle cell disease (SCD).

Sickle cell disease (SCD) is characterized by a prothrombotic state. Plasminogen activator inhibitor-1 (PAI-1) is known to modulate fibrinolysis, lung injury/fibrosis, and angiogenesis. However, its role in SCD is less understood, and the molecular mechanisms underlying increased PAI-1 are unknown. Herein, we show a novel link between PAI-1 and sickle erythropoiesis. Plasma PAI-1 levels were high in SCD patients at steady state and in two humanized sickle mouse models, with increased PAI-1 immunolabeling in sickle mouse lung, bronchial epithelial cells, alveolar macrophages, and pulmonary microvascular endothelial cells. Placenta growth factor (PlGF), released at high levels by sickle erythroblasts, induced PAI-1 expression in primary human pulmonary microvascular endothelial cells and monocytes through activation of c-Jun N-terminal kinase (JNK), NADPH oxidase, and hypoxia-inducible factor-1α (HIF-1α). Analysis of the human PAI-1 promoter revealed this induction was mediated by hypoxia-response element (HRE)-1, HRE-2, and distal activator protein (AP-1) sites. We also identify the involvement of c-Jun, c-Jun/c-Fos, and JunD, but not JunB, in binding with AP-1 sites of the PAI-1 promoter upon PlGF induction. Consistent with these findings, levels of PAI-1 were low in PlGF knock-out mice and sickle-PlGF knock-out mice; overexpression of PlGF in normal mice increased circulating PAI-1. In conclusion, we identify a novel mechanism of PAI-1 elevation in SCD.

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 role of maternal CREB in early embryogenesis of Xenopus laevis

In Xenopus embryos, body patterning and cell specification are initiated by transcription factors, which are themselves transcribed during oogenesis, and their mRNAs are stored for use after fertilization. We have previously shown that the T-box transcription factor VegT is both necessary and sufficient to initiate transcription of all endoderm, and most mesoderm genes. In the absence of maternal VegT, no mesodermal organs (including the heart) or endodermal organs form. A second maternal transcription factor XTcf3 acts as a global repressor of transcription of dorsal genes, whose repression is inactivated on the dorsal side by a maternally encoded Wnt signaling pathway. In the absence of beta-catenin, no mesodermal or endodermal organs form. We show here that the maternally encoded transcription factor CREB is also essential for development. It is required for the initiation of expression of several mesodermal genes, including Xbra, Xcad2, and -3 and also regulates the cardiogenic gene Nkx 2-5. We show that maternal CREB-depleted embryos develop gastrulation defects that are rescued by the reintroduction of activated CREB mRNA. We conclude that maternal CREB must be added to the list of essential maternal transcription factors regulating cell specification in the early embryo.

Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells

SUMMARY Although intestinal homeostasis is maintained by intestinal stem cells (ISCs), regeneration is impaired upon aging. Here, we first uncover changes in intestinal architecture, cell number, and cell composition upon aging. Second, we identify a decline in the regenerative capacity of ISCs upon aging because of a decline in canonical Wnt signaling in ISCs. Changes in expression of Wnts are found in stem cells themselves and in their niche, including Paneth cells and mesenchyme. Third, reactivating canonical Wnt signaling enhances the function of both murine and human ISCs and, thus, ameliorates aging-associated phenotypes of ISCs in an organoid assay. Our data demonstrate a role for impaired Wnt signaling in physiological aging of ISCs and further identify potential therapeutic avenues to improve ISC regenerative potential upon aging.

Drug-induced secretory diarrhea: A role for CFTR

Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecular complexes at discreet microdomains at the plasma membrane, and its chloride channel function is regulated spatiotemporally through protein-protein interactions and cAMP/cGMP-mediated signaling. Drugs that perturb CFTR-containing macromolecular complexes in the intestinal epithelium and upregulate intracellular cAMP and/or cGMP levels can hyperactivate the CFTR channel, causing excessive fluid secretion and secretory diarrhea. Inhibition of CFTR chloride-channel activity may represent a novel approach to the management of drug-induced secretory diarrhea.