A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells.

BACKGROUND: Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear. METHODS: Real-time quantitative PCR (RT-qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT-qPCR and western blotting were used to study the effect of ZFX on ß-catenin signaling. RESULTS: We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/ß-catenin signaling, including c-MYC and CCND1 expression. CONCLUSION: The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector ß-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/ß-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/ß-catenin signaling in OVX mice.

The aOECs Facilitate the Neuronal Differentiation of Neural Stem Cells in the Inflammatory Microenvironment Through Up-Regulation of Bioactive Factors and Activation of Wnt3/ß-Catenin Pathway.

The therapeutic application of neural stem cells (NSCs) in the central nerve system (CNS) injury is a promising strategy for combating irreversible neuronal loss. However, a variety of obvious inflammatory responses following nerve injury rapidly create an unfavorable microenvironment for survival and neuronal differentiation of NSCs in lesion area, limiting the efficacy of NSC-based therapy for CNS injury. It remained unknown how to effectively increase the neuronal differentiation efficiency of NSCs through transplantation. Here, we demonstrated that curcumin (CCM)-activated olfactory ensheathing cells (aOECs) effectively promoted neuronal differentiation of NSCs in the activated microglial inflammatory condition, and co-transplantation of aOECs and NSCs improved neurological recovery of rats after spinal cord injury (SCI), as evidenced by higher expression levels of neuronal markers and lower expression levels of glial markers in the differentiated cells, greater number of Tuj-1-positive cells as well as higher Basso, Beattie, and Bresnahan (BBB) locomotor scale, compared to the corresponding controls. Pathologically, hematoxylin and eosin (HE) staining and immunostaining also showed that aOECs remarkably enhanced the in vivo neuronal differentiation of NSCs and migration, and nerve repair. Further analysis revealed that the underlying mechanisms of aOECs potentiating the neuronal conversion of NSCs under inflammatory environment were tightly associated with up-regulation of anti-inflammatory cytokines and neurotrophic factors in OECs, and importantly, the activation of Wnt3/ß-catenin pathway was likely involved in the mechanisms underlying the observed cellular events. Therefore, this study provides a promising strategy for SCI repair by co-transplantation of aOECs and NSCs.

Sequenced Somatic Cell Reprogramming and Differentiation Inside Nested Hydrogel Droplets.

The efficient genesis of pluripotent cells or therapeutic cells for regenerative medicine involves several external manipulations and conditioning protocols, which drives down clinical applicability. Automated programming of the genesis by microscale physical forces and chronological biochemistry can increase clinical success. The design and fabrication of nested polysaccharide droplets (millimeter-sized) with cell sustaining properties of natural tissues and intrinsic properties for time and space evolution of cell transformation signals between somatic cells, pluripotent cells and differentiated therapeutic cells in a swift and efficient manner without the need for laborious external manipulation are reported. Cells transform between phenotypic states by having single and double nested droplets constituted with extracellular matrix proteins and reprogramming, and differentiation factors infused chronologically across the droplet space. The cell transformation into germ layer cells and bone cells is successfully tested in vitro and in vivo and promotes the formation of new bone tissues. Thus, nested droplets with BMP-2 loaded guests synthesize mineralized bone tissue plates along the length of a cranial non-union bone defect at 4 weeks. The advantages of sequenced somatic cell reprogramming and differentiation inside an individual hydrogel module without external manipulation, promoted by formulating tissue mimetic physical, mechanical, and chemical microenvironments are shown.

Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury.

The loss of stem cells residing in the base of the intestinal crypt has a key role in radiation-induced intestinal injury. In particular, Lgr5+ intestinal stem cells (ISCs) are indispensable for intestinal regeneration following exposure to radiation. Mesenchymal stem cells (MSCs) have previously been shown to improve intestinal epithelial repair in a mouse model of radiation injury, and, therefore, it was hypothesized that this protective effect is related to Lgr5+ ISCs. In this study, it was found that, following exposure to radiation, transplantation of MSCs improved the survival of the mice, ameliorated intestinal injury and increased the number of regenerating crypts. Furthermore, there was a significant increase in Lgr5+ ISCs and their daughter cells, including Ki67+ transient amplifying cells, Vil1+ enterocytes and lysozyme+ Paneth cells, in response to treatment with MSCs. Crypts isolated from mice treated with MSCs formed a higher number of and larger enteroids than those from the PBS group. MSC transplantation also reduced the number of apoptotic cells within the small intestine at 6 h post-radiation. Interestingly, Wnt3a and active ß-catenin protein levels were increased in the small intestines of MSC-treated mice. In addition, intravenous delivery of recombinant mouse Wnt3a after radiation reduced damage in the small intestine and was radioprotective, although not to the same degree as MSC treatment. Our results show that MSCs support the growth of endogenous Lgr5+ ISCs, thus promoting repair of the small intestine following exposure to radiation. The molecular mechanism of action mediating this was found to be related to increased activation of the Wnt/ß-catenin signaling pathway.

Anterior-Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-, and BMP-Signaling.

As known from model organisms, such as frog, fish, mouse, and chicken, the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid, TGF-ß-ligands, Wnt-, and BMP-inhibitors in the anterior half of the DE sheet. However, it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut, we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-ß-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut toward a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2, BMP-4, and RA induces the formation of CDX2-positive posterior endoderm. Thus, these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. Stem Cells 2016;34:2635-2647.

Maintenance of Skin Epithelial Stem Cells by Wnt-3a In Vitro.

CD49f+ CD34+ cells, a population rich in skin epithelial stem cells (EpSCs), were obtained from adult mouse skin and cultured with Wnt-3a for 10 days. On day 10, CD49f+ CD34+ cells were sorted and subjected to a second 10-day culture with Wnt-3a. The same procedures were repeated until fifteenth 10-day culture. CD49f+ CD34+ cells obtained from each 10-day culture retained the same EpSC-characteristics as seen in the original EpSCs from adult mouse skin. Here, wedescribe the culture protocol using Wnt-3a for successful maintenance of EpSCs.

Multisystemic Disease Modeling of Liver-Derived Protein Folding Disorders Using Induced Pluripotent Stem Cells (iPSCs).

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.

Wnt signaling regulates homeostasis of the periodontal ligament.

BACKGROUND AND OBJECTIVE: In health, the periodontal ligament maintains a constant width throughout an organism's lifetime. The molecular signals responsible for maintaining homeostatic control over the periodontal ligament are unknown. The purpose of this study was to investigate the role of Wnt signaling in this process by removing an essential chaperone protein, Wntless (Wls), from odontoblasts and cementoblasts, and observing the effects of Wnt depletion on cells of the periodontal complex. MATERIAL AND METHODS: The Wnt responsive status of the periodontal complex was assessed using two strains of Wnt reporter mice: Axin2(LacZ/+) and Lgr5(LacZ/+) . The function of this endogenous Wnt signal was evaluated by conditionally eliminating the Wntless (Wls) gene using an osteocalcin Cre driver. The resulting OCN-Cre;Wls (fl/fl) mice were examined using micro-computed tomography and histology, immunohistochemical analyses for osteopontin, Runx2 and fibromodulin, in-situ hybridization for osterix and alkaline phosphatase activity. RESULTS: The adult periodontal ligament is Wnt responsive. Elimination of Wnt signaling in the periodontal complex of OCN-Cre;Wls(fl/fl) mice resulted in a wider periodontal ligament space. This pathologically increased periodontal width is caused by a reduction in the expression of osteogenic genes and proteins, which results in thinner alveolar bone. A concomitant increase in fibrous tissue occupying the periodontal space was observed, along with a disruption in the orientation of the periodontal ligament. CONCLUSION: The periodontal ligament is a Wnt-dependent tissue. Cells in the periodontal complex are Wnt responsive, and eliminating an essential component of the Wnt signaling network leads to a pathological widening of the periodontal ligament space. Osteogenic stimuli are reduced, and a disorganized fibrillary matrix results from the depletion of Wnt signaling. Collectively, these data underscore the importance of Wnt signaling in homeostasis of the periodontal ligament.

R-spondins are newly recognized players in osteoarthritis that regulate Wnt signaling in osteoblasts.

OBJECTIVE: Clinical and in vitro studies suggest that altered osteogenesis or bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Wnt signaling plays a key role in osteogenesis via the canonical Wnt/ß-catenin signaling pathway. Two of the R-spondins, Rspo-1 and Rspo-2, a family of 4 proteins unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. The purpose of this study was to investigate the potential role of Rspo-1 and Rspo-2 in OA osteoblasts. METHODS: Primary human normal and OA osteoblasts were prepared from tibial plateaus. The expression of Rspo-1 and Rspo-2 was evaluated by quantitative reverse transcription-polymerase chain reaction analysis. Western blot analysis was used to determine Rspo-2, ß-catenin, and phospho-ß-catenin levels. Wnt/ß-catenin signaling was evaluated using the TOPflash T cell factor (TCF)/lymphoid enhancer factor (LEF) luciferase reporter assay. Mineralization was evaluated by alizarin red staining. RESULTS: The expression of Rspo-1 was similar in normal and OA osteoblasts, whereas the expression and production of Rspo-2 were reduced in OA osteoblasts due to elevated levels of transforming growth factor ß1 in these cells. The reduced Wnt-3a-dependent TOPflash TCF/LEF luciferase reporter activity in OA osteoblasts as compared to normal osteoblasts was corrected by the addition of recombinant human Rspo-2. Wnt-3a-dependent ß-catenin levels were also corrected in OA osteoblasts by Rspo-2 addition. Wnt-3a alone increased the mineralization of OA osteoblasts, which was further increased by Rspo-2. CONCLUSION: Reduced Rspo-2 levels in OA osteoblasts are responsible, at least in part, for their reduced Wnt/ß-catenin signaling and abnormal mineralization. As Rspo-2 is a secreted soluble protein, this could lead to potential new avenues of treatment of OA.