Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p<0.05), indicating that non-cycling (G0) cells entered the cell cycle following the doxorubicin exposure. Support for this notion was obtained also in vitro. A pharmacologically relevant dose (1µM) resulted in a marked accumulation of Ki67 positive BE(2)-C cells (p<0.0001), as well as a >3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation.

Loss of miR-514a-3p regulation of PEG3 activates the NF-kappa B pathway in human testicular germ cell tumors.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.

Induction of sensory neurons from neuroepithelial stem cells by the ISX9 small molecule.

Hearing impairment most often involves loss of sensory hair cells and auditory neurons. As this loss is permanent in humans, a cell therapy approach has been suggested to replace damaged cells. It is thus of interest to generate lineage restricted progenitor cells appropriate for cell based therapies. Human long-term self-renewing neuroepithelial stem (lt-NES) cell lines exhibit in vitro a developmental potency to differentiate into CNS neural lineages, and importantly lack this potency in vivo, i.e do not form teratomas. Small-molecules-driven differentiation is today an established route obtain specific cell derivatives from stem cells. In this study, we have investigated the effects of three small molecules SB431542, ISX9 and Metformin to direct differentiation of lt-NES cells into sensory neurons. Exposure of lt-NES cells to Metformin or SB431542 did not induce any marked induction of markers for sensory neurons. However, a four days exposure to the ISX9 small molecule resulted in reduced expression of NeuroD1 mRNA as well as enhanced mRNA levels of GATA3, a marker and important player in auditory neuron specification and development. Subsequent culture in the presence of the neurotrophic factors BDNF and NT3 for another seven days yielded a further increase of mRNA expression for GATA3. This regimen resulted in a frequency of up to 25-30% of cells staining positive for Brn3a/Tuj1. We conclude that an approach with ISX9 small molecule induction of lt-NES cells into auditory like neurons may thus be an attractive route for obtaining safe cell replacement therapy of sensorineural hearing loss.

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.

Characterization of stem-like cells in mucoepidermoid tracheal paediatric tumor.

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.

Experimental teratoma: at the crossroad of fetal- and onco-development.

Xenografting is the so far only available in vivo model for assessing pluripotency of human stem cells. This review describes known biological features of experimental teratoma from human pluripotent stem cells. We focus on the dual nature mimicking both normal and abnormal development, and propose this model system to be particularly interesting for investigations of the relationship between developmentally controlled differentiation and neoplasia of embryonic origin. In resemblance to the wide range of clinical teratomas, pluripotent stem cell (PSC) induced teratoma (PSCT) typically shows a mixture of developing tissues in randomly distributed compartments. The combined literature suggests that for teratomas derived from human diploid bona fide PSC the embryonic development in the separate tissue-niches can show a controlled differentiation into organoid patterns closely mimicking early development. In the experimental situation such PSCT human homologous in vivo tissue-niches have been shown to provide also matching microenvironment for a micrometastatic colonization and outgrowth of embryonic tumors transplanted directly from patients. Single or small clusters of normal and neoplastic cells can easily be visualized together in microscope-based imaging systems, enabling multi-parameter detection of in the scans of tissue slides/specimens.

Glial origin of mesenchymal stem cells in a tooth model system.

Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

Tropism of the in situ growth from biopsies of childhood neuroectodermal tumors following transplantation into experimental teratoma.

Experimental teratoma induced from human pluripotent stem cells with normal karyotype can be described as a failed embryonic process and includes besides advanced organoid development also large elements of tissue with a prolonged occurrence of immature neural components. Such immature components, although benign, exhibit strong morphological resemblance with tumors of embryonic neuroectodermal origin. Here, we demonstrate that biopsy material from childhood tumors of neural embryonic origin transplanted to mature experimental teratoma can show an exclusive preference for matching tissue. Tumor specimens from five children with; Supratentorial primitive neuroectodermal tumor (sPNET); Pilocytic astrocytoma of the brainstem; Classic medulloblastoma; peripheral primitive neuroectodermal tumor (pPNET) or neuroblastoma (NB), respectively, were transplanted. Analysis of up to 120 sections of each tumor revealed an engraftment for three of the transplanted tumors: pPNET, sPNET, and NB, with a protruding growth from the latter two that were selected for detailed examination. The histology revealed a strict tropism with a non-random integration into what morphologically appeared as matched embryonic microenvironment recuperating the patient tumor histology. The findings suggest specific advantages over xenotransplantation and lead us to propose that transplantation to the human embryonic microenvironment in experimental teratoma can be a well-needed complement for preclinical in vivo studies of childhood neuroectodermal tumors.

Nodal expression and detection in cancer: experience and challenges.

Nodal is a TGF-ß-related embryonic morphogen that is expressed in multiple human cancers. Detection of Nodal expression in these tissues can be challenging if issues related to Nodal transcription and protein processing are not considered. Here, we discuss certain characteristics related to Nodal expression and function and how these can facilitate acquisition and interpretation of expression data, contributing to our understanding of the potential role of Nodal in human cancer. We also discuss how Nodal could be exploited clinically as a novel biomarker for cancer progression and therapeutic target.

Concise review: Cord blood banking, transplantation and induced pluripotent stem cell: success and opportunities.

Hematopoietic cell transplantation (HCT) has become a standard practice to treat a number of malignant and nonmalignant hematologic diseases. Bone marrow, mobilized peripheral blood, and umbilical cord blood can all serve as primary sources of cells for HCT. The number of cord blood units currently stored is large, although it represents only a fraction of potential collections. With much of the collection being sequestered in private banks for possible autologous use, there is a reason to expect that public banks may not be able to provide for the demand in coming years as use of cord blood for treatment of patients with diseases such as leukemia and lymphoma continues to increase. We suggest that a possible solution to encourage private banks to share their valuable units is to apply recent methodologies to generate induced pluripotent stem cells from cord cells and to optimize techniques to generate hematopoietic lineages from them. This strategy would allow us to take advantage of the units already collected under appropriate regulatory guidelines, to access a pristine cell that can be converted to a pluripotent cell at a much higher efficiency and in a shorter time period than other cells. The ability to potentially replenish a used cord unit with new cells, as well as extend the potential utility of cord blood for additional therapeutic applications, should allow banks to develop an appropriate business model for both private and public cord blood banks to flourish.

Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

Injection of human mesenchymal stem cells improves healing of vocal folds after scar excision--a xenograft analysis.

OBJECTIVES: Using a xenograft model the aim was to analyze if injection of human mesenchymal stem cells (hMSC) into the rabbit vocal fold (VF), after excision of an established scar, can improve the functional healing of the VF. STUDY DESIGN: Prospective design with an experimental xenograft model. METHODS: The VFs of 12 New Zealand rabbits were injured by a bilateral localized resection. After 9 weeks the scar after the resection was excised and hMSC were injected into the VFs. After another 10 weeks 10 VFs were dissected and stained for histology. Lamina propria thickness and relative content of collagen type I were measured. Viscoelasticity of 14 VFs at phonatory frequencies was quantified by a simple-shear rheometer. The hMSC survival was determined using a human DNA specific reference probe, that is, FISH analysis. RESULTS: The viscoelastic measurements, that is, dynamic viscosity and elastic shear modulus for the hMSC-treated VFs, were found to be similar to those of normal controls and were significantly lower than those of untreated controls (P < .05). A significant reduction in lamina propria thickness was also shown for the hMSC treated VFs compared with the untreated VFs (P < .05). This histologic finding corresponded with the viscoelastic results. No hMSC survived 10 weeks after the injection. CONCLUSIONS: Human mesenchymal stem cells injected into the rabbit VF following the excision of a chronic scar, were found to enhance the functional healing of the VF with reduced lamina propria thickness and restored viscoelastic shear properties.

Injection of human mesenchymal stem cells improves healing of scarred vocal folds: analysis using a xenograft model.

OBJECTIVES/HYPOTHESIS: The aims were to analyze if improved histological and viscoelastic properties seen after injection of human mesenchymal stem cells (hMSCs) in scarred vocal folds (VFs) of rabbits are sustainable and if the injected hMSCs survive 3 months in the VFs. STUDY DESIGN: Experimental xenograft model. METHODS: Eighteen VFs of 11 New Zealand white rabbits were scarred by a bilateral localized resection. After 3 months the animals were sacrificed. Twelve VFs were dissected and stained for histology, lamina propria thickness, and relative collagen type I analyses. The hMSCs survival was analyzed using a human DNA-specific reference probe, that is, fluorescence in situ hybridization staining. Viscoelasticity, measured as the dynamic viscosity and elastic modulus, was analyzed in a parallel-plate rheometer for 10 VFs. RESULTS: The dynamic viscosity and elastic modulus of hMSC-treated VFs were similar to that of normal controls and significantly improved compared to untreated controls (P < .05). A reduction in lamina propria thickness and relative collagen type 1 content were also shown for the hMSC-treated VFs compared to the untreated VFs (P < .05). The histological pictures corresponded well to the viscoelastic results. No hMSCs survived. CONCLUSIONS: Human mesenchymal stem cells injected into a scarred vocal fold of rabbit enhance healing of the vocal fold with reduced lamina propria thickness and collagen type I content and restore the viscoelastic function.

Lineage-specific promoter DNA methylation patterns segregate adult progenitor cell types.

Mesenchymal stem cells (MSCs) can differentiate into multiple mesodermal cell types in vitro; however, their differentiation capacity is influenced by their tissue of origin. To what extent epigenetic information on promoters of lineage-specification genes in human progenitors influences transcriptional activation and differentiation potential remains unclear. We produced bisulfite sequencing maps of DNA methylation in adipogenic, myogenic, and endothelial promoters in relation to gene expression and differentiation capacity, and unravel a similarity in DNA methylation profiles between MSCs isolated from human adipose tissue, bone marrow (BM), and muscle. This similarity is irrespective of promoter CpG content. Methylation patterns of MSCs are distinct from those of hematopoietic progenitor cells (HPCs), pluripotent human embryonic stem cells (hESCs), and multipotent hESC-derived mesenchymal cells (MCs). Moreover, in vitro MSC differentiation does not affect lineage-specific promoter methylation states, arguing that these methylation patterns in differentiated cells are already established at the progenitor stage. Further, we find a correlation between lineage-specific promoter hypermethylation and lack of differentiation capacity toward that lineage, but no relationship between weak promoter methylation and capacity of transcriptional activation or differentiation. Thus, only part of the restriction in differentiation capacity of tissue-specific stem cells is programmed by promoter DNA methylation: hypermethylation seems to constitute a barrier to differentiation, however, no or weak methylation has no predictive value for differentiation potential.

Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells.

Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indefinite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding and differentiating human embryonic stem cells (ESCs) might not be directly applicable to human iPSCs. However, we show here that human iPSCs rely on activin/nodal signaling to control Nanog expression and thereby maintain pluripotency, thus revealing their mechanistic similarity to human ESCs. We also show that growth factors necessary and sufficient for achieving specification of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically defined medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent.

Species-specific in vivo engraftment of the human BL melanoma cell line results in an invasive dedifferentiated phenotype not present in xenografts.

For clinically relevant studies on melanoma progression and invasiveness, in vivo experimental systems with a human cellular microenvironment would be advantageous. We have compared tumor formation from a human cutaneous malignant melanoma cell line (BL), after injection as conventional xenografts in the mouse, or when injected into a predominantly species-specific environment of human embryonic stem cell-derived teratoma induced in the mouse (the hEST model). The resulting melanoma histology was generally analogous, both systems showing delimited densely packed areas with pleomorphic cells of malignant appearance. A specificity of the integration process into the human embryonic teratoma tissues was indicated by the melanoma exclusively being found in areas compatible with condensed mesenchyme, similar to neural crest development. Here, also enhanced neovascularization was seen within the human mesenchymal tissues facing the BL melanoma growth. Furthermore, in the hEST model an additional melanoma cell phenotype occurred, located at the border of, or infiltrating into, the surrounding human loose mesenchymal fibrous stroma. This BL population had a desmoplastic spindle-like appearance, with markers indicative of dedifferentiation and migration. The appearance of this apparently more aggressive phenotype, as well as the induction of human angiogenesis, shows specific interactions with the human embryonic microenvironment in the hEST model. In conclusion, these data provide exciting options for using the hEST model in molecular in vivo studies on differentiation, invasiveness, and malignancy of human melanoma, while analyzing species-specific reactions in vivo.

Differentiation of human embryonic stem cells into osteogenic or hematopoietic lineages: a dose-dependent effect of osterix over-expression.

Enhanced differentiation of human embryonic stem cells (HESCs), induced by genetic modification could potentially generate a vast number of diverse cell types. Such genetic modifications have frequently been achieved by over-expression of individual regulatory proteins. However, careful evaluation of the expression levels is critical, since this might have important implications for the differentiation potential of HESCs. To date, attempts to promote osteogenesis by means of gene transfer into HESCs using the early bone "master" transcription factor osterix (Osx) have not been reported. In this study, we attained HESC subpopulations expressing two significantly different levels of Osx, following lentiviral gene transfer. Both subpopulations exhibited spontaneous differentiation and reduced expression of markers characteristic of the pluripotent phenotype, such as SSEA3, Tra1-60, and Nanog, In order to promote bone differentiation, the cells were treated with ascorbic acid, beta-glycerophosphate and dexamethasone. The high level of Osx, compared to endogenous levels found in primary human osteoblasts, did not enhance osteogenic differentiation, and did not up-regulate collagen I expression. We show that the high Osx levels instead induced the commitment towards the hematopoietic-endothelial lineage-by up-regulating the expression of CD34 and Gata1. However, low levels of Osx up-regulated collagen I, bone sialoprotein and osteocalcin. Conversely, forced high level expression of the homeobox transcription factor HoxB4, a known regulator for early hematopoiesis, promoted osteogenesis in HESCs, while low levels of HoxB4 lead to hematopoietic gene expression.

Isolation and production of cells suitable for human therapy: challenges ahead.

Considerable practical hurdles must be overcome prior to the broad application of stem cell therapies. We outline challenges that may vary across different models of cell therapy, including the following broad concepts: issues related to the sourcing of material, and issues related to product manufacturing, shipping, storage and tracking, and standardization.