Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules α6-integrin, αv-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for α6-integrin, and negative or low αv-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Patterns of Stem Cell Divisions Contribute to Plant Longevity

The lifespan of plants ranges from a few weeks in annuals to thousands of years in trees. It is hard to explain such extreme longevity considering that DNA replication errors inevitably cause mutations. Without purging through meiotic recombination, the accumulation of somatic mutations will eventually result in mutational meltdown, a phenomenon known as Muller’s ratchet. Nevertheless, the lifespan of trees is limited more often by incidental disease or structural damage than by genetic aging. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves and grow out to form branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Here, we use stem cell ablation and quantitative cell-lineage analysis to show that axillary meristems are set aside early, analogous to the metazoan germline. While neighboring cells divide vigorously, axillary meristem precursors maintain a quiescent state, with only 7–9 cell divisions occurring between the apical and axillary meristem. During iterative branching, the number of branches increases exponentially, while the number of cell divisions increases linearly. Moreover, computational modeling shows that stem cell arrangement and positioning of axillary meristems distribute somatic mutations around the main shoot, preventing their fixation and maximizing genetic heterogeneity. These features slow down Muller’s ratchet and thereby extend lifespan.

Autologous hemopoietic stem cell transplantation in severe rheumatoid arthritis: A report from the EBMT and ABMTR

Objective. Since 1996, autologous hemopoietic stem cell transplantation (HSCT) has been used to treat severe rheumatoid arthritis (RA). To date, published reports have been individual cases or series containing small numbers. This study combined the worldwide experience in a single analysis. Methods. The Autoimmune Disease Databases of the European Group for Blood and Marrow Transplantation (EBMT) and the Autologous Blood and Marrow Transplant Registry (ABMTR) were used to identify patients with RA treated with autologous HSCT. Further information relating to patient and treatment-specific variables was obtained by questionnaire. Results. Seventy-six patients were registered from 15 centers. Seventy-three patients had received autologous HSCT, and in 3 patients hematopoietic stem cells (HSC) were mobilized but not transplanted. Transplanted patients (median age 42 yrs, 74% female, 86% rheumatoid factor positive) had been previously treated with a mean of 5 (range 2-9) disease modifying antirheumatic drugs (DMARD). Significant functional impairment was present, with a median Health Assessment Questionnaire (HAQ) score of 1.4 (range 1.1-2.0) and Steinbrocker score mean 2.39 (SD 0.58). The high dose treatment regimen was cyclophosphamide (CYC) alone in the majority of patients, mostly 200 mg/kg (n = 62). Seven patients received anti-thymocyte globulin (ATG) in addition to CYC, 2 patients busulfan and CYC (BuCYC), and one patient CYC with total body irradiation and ATG. One patient received fludarabine with ATG. Following treatment, one patient received bone marrow but the rest received chemotherapy and/or granulocyte colony-stimulating factor mobilized peripheral blood stem cells. The harvest was unmanipulated in 28 patients, the rest receiving some form of lymphocyte depletion, mostly through CD34+ selection. Median followup was 16 months (range 3-55). Responses were measured using the American College of Rheumatology (ACR) criteria. Forty-nine patients (67%) achieved at least ACR 50% response at some point following transplant. There was a significant reduction in the level of disability measured by the HAQ (p < 0.005). Most patients restarted DMARD within 6 months for persistent or recurrent disease activity, which provided disease control in about half the cases. Response was significantly related to seronegative RA (p = 0.02) but not to duration of disease, number of previous DMARD, presence of HLA-DR4, or removal of lymphocytes from the graft. There was no direct transplant related mortality, although one patient, treated with the BuCYC regimen, died 5 months post-transplant from infection and incidental non-small cell lung cancer. Conclusion. Autologous HSCT is a relatively safe form of salvage treatment in severe, resistant RA. In these open label studies significant responses were achieved in most patients, with over 50% achieving an ACR 50 or more response at 12 months. Although the procedure is not curative, recurrent or persistent disease activity may be subsequently controlled in some patients with DMARD. Clinical trials are necessary to develop this approach inpatients with aggressive disease who have failed conventional treatment including anti-tumor necrosis factor agents.

Aβ(1-42) induced metabolic effects in a stem cell derived neuron and astrocyte network.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.

Amyloid β 1-42 induces hypometabolism in human stem cell-derived neuron and astrocyte networks

Alzheimer's disease (AD) is the most common form of dementia, affecting more than 35 million people worldwide. Brain hypometabolism is a major feature of AD, appearing decades before cognitive decline and pathologic lesions. To date, the majority of studies on hypometabolism in AD have used transgenic animal models or imaging studies of the human brain. As it is almost impossible to validate these findings using human tissue, alternative models are required. In this study, we show that human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose, pyruvate, lactate, and glutamate. In addition, a significant increase in the glycogen content of cells was also observed. These changes were accompanied by changes in NAD+ /NADH, ATP, and glutathione levels, suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. Further research using this model may elucidate the mechanisms associated with Aβ-induced hypometabolism.

Nano-silver treatments reduced bacterial colonization and biofilm formation at the stem-ends of cut gladiolus ‘Eerde’ spikes

Postharvest treatments with nano-silver (NS) alleviate bacteria-related stem blockage of some cut flowers to extend their longevity. Gladiolus (Gladiolus hybridus) is a commercially important cut flower species. For the first time, the effects of NS pulses on cut gladiolus ‘Eerde’ spikes were investigated towards reducing bacterial colonization of and biofilm formation on their stems. As compared with a deionized water (DIW) control, pulse treatments with NS at 10, 25 and 50 mg L−1 for 24 h significantly (P ≤ 0.05) prolonged the vase life of cut gladiolus spikes moved into vases containing DIW. The NS treatments enhanced floret ‘opening rate’ and ‘daily ornamental value’. Although there were no significant differences among NS treatments, a 25 mg L−1 NS pulse treatment tended to give the longest vase life and the best ‘display quality’. All NS pulse treatments significantly improved water uptake by and reduced water loss from flowering spikes, thereby delaying the loss of water balance and maintaining relative fresh weight. Fifty (50) mg L−1 NS pulse-treated cut gladiolus spikes tended to exhibit the most water uptake and highest water balance over the vase period. However, there was no significant difference between 25 and 50 mg L−1 NS pulse treatments. Observations of stem-end bacterial proliferation during the vase period on cut gladiolus spikes either with or without NS pulse treatments were performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). As compared to the control treatment, they revealed that the 25 mg L−1 NS pulse treatment effectively inhibited bacterial colonization and biofilm formation on the stem-end cut surface and in the xylem vessels, respectively. In vitro culture of the bacterial microflora and analysis of biofilm architecture using CLSM revealed that NS treatment restricted bacterial biofilm formation. After static culture for 24 h at 35 °C with 25 mg L−1 NS in the medium, no biofilm form or structure was evident. Rather, only limited bacterial cell number and scanty extracellular polysaccharide (EPS) material were observed. In contrast, mature bacterial biofilm architecture comprised of abundant bacteria interwoven with EPS formed in the absence of NS.

Metastability and dynamics of stem cells: from direct observations to inference at the single cell level

Organismal development, homeostasis, and pathology are rooted in inherently probabilistic events. From gene expression to cellular differentiation, rates and likelihoods shape the form and function of biology. Processes ranging from growth to cancer homeostasis to reprogramming of stem cells all require transitions between distinct phenotypic states, and these occur at defined rates. Therefore, measuring the fidelity and dynamics with which such transitions occur is central to understanding natural biological phenomena and is critical for therapeutic interventions.

While these processes may produce robust population-level behaviors, decisions are made by individual cells. In certain circumstances, these minuscule computing units effectively roll dice to determine their fate. And while the 'omics' era has provided vast amounts of data on what these populations are doing en masse, the behaviors of the underlying units of these processes get washed out in averages.

Therefore, in order to understand the behavior of a sample of cells, it is critical to reveal how its underlying components, or mixture of cells in distinct states, each contribute to the overall phenotype. As such, we must first define what states exist in the population, determine what controls the stability of these states, and measure in high dimensionality the dynamics with which these cells transition between states.

To address a specific example of this general problem, we investigate the heterogeneity and dynamics of mouse embryonic stem cells (mESCs). While a number of reports have identified particular genes in ES cells that switch between 'high' and 'low' metastable expression states in culture, it remains unclear how levels of many of these regulators combine to form states in transcriptional space. Using a method called single molecule mRNA fluorescent in situ hybridization (smFISH), we quantitatively measure and fit distributions of core pluripotency regulators in single cells, identifying a wide range of variabilities between genes, but each explained by a simple model of bursty transcription. From this data, we also observed that strongly bimodal genes appear to be co-expressed, effectively limiting the occupancy of transcriptional space to two primary states across genes studied here. However, these states also appear punctuated by the conditional expression of the most highly variable genes, potentially defining smaller substates of pluripotency.

Having defined the transcriptional states, we next asked what might control their stability or persistence. Surprisingly, we found that DNA methylation, a mark normally associated with irreversible developmental progression, was itself differentially regulated between these two primary states. Furthermore, both acute or chronic inhibition of DNA methyltransferase activity led to reduced heterogeneity among the population, suggesting that metastability can be modulated by this strong epigenetic mark.

Finally, because understanding the dynamics of state transitions is fundamental to a variety of biological problems, we sought to develop a high-throughput method for the identification of cellular trajectories without the need for cell-line engineering. We achieved this by combining cell-lineage information gathered from time-lapse microscopy with endpoint smFISH for measurements of final expression states. Applying a simple mathematical framework to these lineage-tree associated expression states enables the inference of dynamic transitions. We apply our novel approach in order to infer temporal sequences of events, quantitative switching rates, and network topology among a set of ESC states.

Taken together, we identify distinct expression states in ES cells, gain fundamental insight into how a strong epigenetic modifier enforces the stability of these states, and develop and apply a new method for the identification of cellular trajectories using scalable in situ readouts of cellular state.

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016