Reprogramming identifies functionally distinct stages of clonal evolution in myelodysplastic syndromes.

Myeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells (HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.

Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.

Altered glycosylation in pancreatic cancer and beyond.

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers and is projected to soon be the second leading cause of cancer death. Median survival of PDA patients is 6-10 mo, with the majority of diagnoses occurring at later, metastatic stages that are refractory to treatment and accompanied by worsening prognoses. Glycosylation is one of the most common types of post-translational modifications. The complex landscape of glycosylation produces an extensive repertoire of glycan moieties, glycoproteins, and glycolipids, thus adding a dynamic and tunable level of intra- and intercellular signaling regulation. Aberrant glycosylation is a feature of cancer progression and influences a broad range of signaling pathways to promote disease onset and progression. However, despite being so common, the functional consequences of altered glycosylation and their potential as therapeutic targets remain poorly understood and vastly understudied in the context of PDA. In this review, the functionality of glycans as they contribute to hallmarks of PDA are highlighted as active regulators of disease onset, tumor progression, metastatic capability, therapeutic resistance, and remodeling of the tumor immune microenvironment. A deeper understanding of the functional consequences of altered glycosylation will facilitate future hypothesis-driven studies and identify novel therapeutic strategies in PDA.

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro.

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Cells with surface expression of CD133highCD71low are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas.

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2-4month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133(high)CD71(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133(high)CD71(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133(high)CD71(low) cells gave rise to colonies that contained duct, acinar, and Insulin(+)Glucagon(+) double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin(+)Glucagon(-) cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133(high)CD71(low) cells. These CD133(high)CD71(low) cells will enable future studies of putative adult pancreas stem cells in vivo.

Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.

Postnatal pancreas is a potential source for progenitor cells to generate endocrine ß-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for ß-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional ß-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133(high) fraction and among 230 micro-manipulated single CD133(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of ß-like cells in vitro.

Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed "Dark" colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133⁺CD49f(low)CD107b(low) phenotype, while pancreatic CFU-Dark are CD133⁻. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.