Jonathan C. Howell

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.

Generating human intestinal tissue from pluripotent stem cells in vitro

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2+SOX17+ endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2+ mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1–3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.

Generating intestinal tissue from stem cells: potential for research and therapy.

Intestinal resection and malformations in adult and pediatric patients result in devastating consequences. Unfortunately, allogeneic transplantation of intestinal tissue into patients has not been met with the same measure of success as the transplantation of other organs. Attempts to engineer intestinal tissue in vitro include disaggregation of adult rat intestine into subunits called organoids, harvesting native adult stem cells from mouse intestine and spontaneous generation of intestinal tissue from embryoid bodies. Recently, by utilizing principles gained from the study of developmental biology, human pluripotent stem cells have been demonstrated to be capable of directed differentiation into intestinal tissue in vitro. Pluripotent stem cells offer a unique and promising means to generate intestinal tissue for the purposes of modeling intestinal disease, understanding embryonic development and providing a source of material for therapeutic transplantation.

Late endocrine effects of childhood cancer

The cure rate for paediatric malignancies is increasing, and most patients who have cancer during childhood survive and enter adulthood. Surveillance for late endocrine effects after childhood cancer is required to ensure early diagnosis and treatment and to optimize physical, cognitive and psychosocial health. The degree of risk of endocrine deficiency is related to the childs sex and their age at the time the tumour is diagnosed, as well as to tumour location and characteristics and the therapies used (surgery, chemotherapy or radiation therapy). Potential endocrine problems can include growth hormone deficiency, hypothyroidism (primary or central), adrenocorticotropin deficiency, hyperprolactinaemia, precocious puberty, hypogonadism (primary or central), altered fertility and/or sexual function, low BMD, the metabolic syndrome and hypothalamic obesity. Optimal endocrine care for survivors of childhood cancer should be delivered in a multidisciplinary setting, providing continuity from acute cancer treatment to long-term follow-up of late endocrine effects throughout the lifespan. Endocrine therapies are important to improve long-term quality of life for survivors of childhood cancer.

Vitamin D Deficiency and Survival in Children after Hematopoietic Stem Cell Transplant.

Vitamin D has endocrine function as a key regulator of calcium absorption and bone homeostasis and also has intracrine function as an immunomodulator. Vitamin D deficiency before hematopoietic stem cell transplantation (HSCT) has been variably associated with higher risks of graft-versus-host disease (GVHD) and mortality. Children are at particular risk of growth impairment and bony abnormalities in the face of prolonged deficiency. There are few longitudinal studies of vitamin D deficient children receiving HSCT, and the prevalence and consequences of vitamin D deficiency 100 days after transplant has been poorly studied. Serum samples from 134 consecutive HSCT patients prospectively enrolled into an HSCT sample repository were tested for 25-hydroxy (25 OH) vitamin D levels before starting HSCT (baseline) and at 100 days after transplantation. Ninety-four of 134 patients (70%) had a vitamin D level < 30 ng/mL before HSCT, despite supplemental therapy in 16% of subjects. Post-transplant samples were available in 129 patients who survived to day 100 post-transplant. Vitamin D deficiency persisted in 66 of 87 patients (76%) who were already deficient before HSCT. Moreover, 24 patients with normal vitamin D levels before HSCT were vitamin D deficient by day 100. Overall, 68% of patients were vitamin D deficient (<30 ng/mL) at day 100, and one third of these cases had severe vitamin D deficiency (<20 ng/mL). Low vitamin D levels before HSCT were not associated with subsequent acute or chronic GVHD, contrary to some prior reports. However, severe vitamin D deficiency (<20 ng/mL) at 100 days post-HSCT was associated with decreased overall survival after transplantation (P = .044, 1-year rate of overall survival: 70% versus 84.1%). We conclude that all pediatric transplant recipients should be screened for vitamin D deficiency before HSCT and at day 100 post-transplant and that aggressive supplementation is needed to maintain sufficient levels.

Poor growth, thyroid dysfunction and vitamin D deficiency remain prevalent despite reduced intensity chemotherapy for hematopoietic stem cell transplantation in children and young adults

Myeloablative conditioning regimens for hematopoietic stem cell transplant (HSCT) are known to affect endocrine function, but little is known regarding reduced intensity conditioning (RIC) regimens. We retrospectively reviewed 114 children and young adults after single RIC HSCT. The analysis was grouped by age (<2 and ⩾2 years) and diagnosis (hemophagocytic lymphohistiocystosis/X-linked lymphoproliferative syndrome (HLH/XLP), other immune disorders, metabolic/genetic disorders). All groups displayed short stature by mean height-adjusted Z-score (HAZ) before (−1.29) and after HSCT (HAZ −1.38, P=0.47). After HSCT, younger children with HLH/XLP grew better (HAZ −3.41 vs −1.65, P=0.006), whereas older subjects had decline in growth (HAZ −0.8 vs −1.01, P=0.06). Those with steroid therapy beyond standard GVHD prophylaxis were shorter than those without (P 0.04). After HSCT, older subjects with HLH/XLP became thinner with a mean body mass index (BMI) Z-score of 1.20 vs 0.64, P=0.02, and similar to metabolic/genetic disorders (BMI-Z= 0.59 vs −0.99, P<0.001). BMI increased among younger children in these same groups. Thyroid function was abnormal in 24% (18/76). 25-OH vitamin D levels were insufficient in 73% (49/65), with low bone mineral density in 8 of 19 evaluable subjects. Despite RIC, children and young adults still have significant late endocrine effects. Further research is required to compare post-transplant endocrine effects after RIC to those after standard chemotherapy protocols.

Growth hormone improves short stature in children with Diamond-Blackfan anemia

Diamond‐Blackfan anemia (DBA), an inherited marrow failure syndrome, has severe hypoplastic anemia in infancy and association with aplastic anemia, MDS/leukemia, and other malignancies. Short stature is present in most patients. Isolated cases have demonstrated improved growth on growth hormone (GH) therapy.

Single Ultra-High-Dose Cholecalciferol to Prevent Vitamin D Deficiency in Pediatric Hematopoietic Stem Cell Transplantation

Vitamin D deficiency is prevalent among childhood hematopoietic stem cell transplantation (HSCT) recipients and associated with inferior survival at 100 days after transplantation. Achieving and maintaining therapeutic vitamin D levels in HSCT recipients is extremely challenging in the first 3 to 6 months after transplantation due to poor compliance in the setting of mucositis and the concomitant use of critical transplantation drugs that interfere with vitamin D absorption. We sought to evaluate the safety and efficacy of a single, ultra-high-dose of vitamin D given before childhood HSCT to maintain levels in a therapeutic range during the peritransplantation period. Ten HSCT recipients with pretransplantation 25-OH vitamin D (25OHD) level <50 ng/mL and with no history of hypercalcemia, nephrolithiasis, or pathological fractures were enrolled on this pilot study. A single enteral vitamin D dose (maximum 600,000 IU) was administered to each patient based on weight and pretransplantation vitamin D level before the day of HSCT. Vitamin D levels between 30 and 150 ng/mL were considered therapeutic. All patients received close clinical observation and monitoring of 25OHD levels, calcium, phosphate, parathyroid hormone, urine calcium/creatinine ratio, and n-telopeptide for safety and efficacy assessment. The mean age of the study subjects was 5.8 ± 4.9 years, and the mean pretransplantation 25OHD level was 28.9 ± 13.1 ng/mL. All patients tolerated single, ultra-high-oral dose of vitamin D under direct medical supervision. No other oral vitamin D supplements were administered during the observation window of 8 weeks. Three of 10 patients received 400 IU/day of vitamin D in parenteral nutrition only for 5 days during the study window. A mean peak serum vitamin D level of 80.4 ± 28.6 ng/mL was reached at a median of 9 days after the vitamin D dose. All patients achieved a therapeutic vitamin D level of >30 ng/mL. Mean vitamin D levels were sustained at or above 30 ng/mL during the 8-week observation window. There were no electrolyte abnormalities attributed to the ultra-high-dose of vitamin D. Most patients had mildly elevated urine calcium/creatinine ratios during treatment, but none showed clinical or radiologic signs of nephrocalcinosis or nephrolithiasis. Our findings indicate that single ultra-high-oral dose vitamin D treatment given just before HSCT is safe and well tolerated in the immediate peritransplant period in children. Patients in our study were able to achieve and sustain therapeutic vitamin D levels throughout the critical period during which vitamin D insufficiency is associated with decreased overall survival. Larger prospective studies are needed to address the impact of single ultra-high-dose vitamin D treatment on HSCT outcomes.