generation-maintenance-characterization-human-pluripotent-stem-cell

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Medical University of South Carolina

The Eph receptor tyrosine kinase family member EphA2 plays a pivotal role in modulating cytoskeletal dynamics to control cancer cell motility and invasion. EphA2 is frequently upregulated in diverse solid tumors and has emerged as a viable druggable target. We previously reported that extracellular Hsp90 (eHsp90), a known pro-motility and invasive factor, collaborates with EphA2 to regulate tumor invasion in the absence of its cognate ephrin ligand. Here, we aimed to further define the molecular and functional relationship between EphA2 and eHsp90. Ligand dependent ephrin A1 signaling promotes RhoA activation and altered cell morphology to favor transient cell rounding, retraction, and diminished adhesion. Exposure of EphA2-expressing cancer cells to ligand herein revealed a unique role for eHsp90 as an effector of cytoskeletal remodeling. Notably, blockade of eHsp90 via either neutralizing antibodies or administration of cell-impermeable Hsp90-targeted small molecules significantly attenuated ligand dependent cell rounding in diverse tumor types. Although eHsp90 blockade did not appear to influence receptor internalization, downstream signaling events were augmented. In particular, eHsp90 activated a Src-RhoA axis to enhance ligand dependent cell rounding, retraction, and ECM detachment. Moreover, eHsp90 signaling via this axis stimulated activation of the myosin pathway, culminating in formation of an EphA2-myosin complex. Inhibition of either eHsp90 or Src was sufficient to impair ephrin A1 mediated Rho activation, activation of myosin intermediates, and EphA2-myosin complex formation. Collectively, our data support a paradigm whereby eHsp90 and EphA2 exhibit molecular crosstalk and functional cooperation within a ligand dependent context to orchestrate cytoskeletal events controlling cell morphology and attachment.

Molecular and functional crosstalk between extracellular Hsp90 and ephrin A1 signaling.

Epigenetic alterations comprise key regulatory events that dynamically alter gene expression and their deregulation is commonly linked to the pathogenesis of various diseases, including cancer. Unlike DNA mutations, epigenetic alterations involve modifications to proteins and nucleic acids that regulate chromatin structure without affecting the underlying DNA sequence, altering the accessibility of the transcriptional machinery to the DNA, thus modulating gene expression. In cancer cells, this often involves the silencing of tumor suppressor genes or the increased expression of genes involved in oncogenesis. Advances in laboratory medicine have made it possible to map critical epigenetic events, including histone modifications and DNA methylation, on a genome-wide scale. Like the identification of genetic mutations, mapping of changes to the epigenetic landscape has increased our understanding of cancer progression. However, in contrast to irreversible genetic mutations, epigenetic modifications are flexible and dynamic, thereby making them promising therapeutic targets. Ongoing studies are evaluating the use of epigenetic drugs in chemotherapy sensitization and immune system modulation. With the preclinical success of drugs that modify epigenetics, along with the FDA approval of epigenetic drugs including the DNA methyltransferase 1 (DNMT1) inhibitor 5-azacitidine and the histone deacetylase (HDAC) inhibitor vorinostat, there has been a rise in the number of drugs that target epigenetic modulators over recent years.

Targeting Chromatin Remodeling for Cancer Therapy.

In recent years, advances in human pluripotent stem cell (hPSC) biology have enabled the generation of gastrointestinal (GI) organoids which recapitulate aspects of normal organ development. HPSC derived gastrointestinal organoids are comprised of epithelium and mesenchyme and have a remarkable ability to self-organize and recapitulate early stages of human intestinal development. Furthermore, hPSC derived organoids can be transplanted into immunocompromised mice which allows further maturation of both the epithelium and mesenchyme. In this review, we will briefly summarize work from model systems which has elucidated mechanisms of GI patterning and how these insights have been used to guide the differentiation of hPSCs into organoids resembling small intestine and colon. We will succinctly discuss how developmental principles have been used to promote maturation of human intestinal organoids (HIOs)  as well as to introduce an enteric nervous system into HIOs. We will then concisely review how organoids have been used to study human pathogens, how new genetic and bioengineering tools are being applied to organoid research, and how this integration has allowed researchers to elucidate mechanisms of human development and disease. Finally, we will briefly discuss remaining challenges in the field and how they can be addressed. HPSC derived organoids are promising new model systems which hold the potential of unlocking unknown mechanisms of human gastrointestinal development and disease.

Insights Into Human Development and Disease From Human Pluripotent Stem Cell Derived Intestinal Organoids.

Advances in human pluripotent stem cell (hPSC) biology now allow the generation of organoids that resemble different regions of the gastrointestinal tract. Generation of region-specific organoids has been facilitated by developmental biology studies carried out in model organisms such as mouse, frog and chick. By mimicking embryonic development, hPSC-derived human colonic organoids (HCOs) can be generated through a stepwise differentiation, first into definitive endoderm (DE), then into mid/hindgut spheroids which are then patterned into posterior gut tissue which gives rise to HCOs following prolonged in vitro culture. HCOs undergo transitions similar to those observed in the developing colon of model organisms and human embryos. HCOs develop into tissue that resembles fetal colon on the basis of morphology, gene expression and presence of differentiated cell types. Generation of HCOs without the proper training or expertise can be a daunting task. Here, we describe a detailed protocol for differentiating hPSCs into HCOs, we include suggestions for troubleshooting these differentiations, and we discuss experimental design considerations. We have also highlighted the key advantages and limitations of the system.

Abdelkader Daoud

Department of Regenerative Medicine and Cell Biology

Medical University of South Carolina

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Abdelkader Daoud

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Regenerative Medicine and Cell Biology

Medical University of South Carolina

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Department of Regenerative Medicine and Cell Biology