In vitro

In vitro (meaning in glass, or in the glass) studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology and its subdisciplines are traditionally done in labware such as test tubes, flasks, Petri dishes, and microtiter plates. Studies conducted using components of an organism that have been isolated from their usual biological surroundings permit a more detailed or more convenient analysis than can be done with whole organisms; however, results obtained from in vitro experiments may not fully or accurately predict the effects on a whole organism. In contrast to in vitro experiments, in vivo studies are those conducted in living organisms, including humans, known as clinical trials, and whole plants. Definition In vitro (in glass; often not italicized in English usage) studies are conducted using components of an organism that have been isolated from
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Related publications (100)

Muscle stem cell's in vitro bipolar niche

Adrien de Tonnac

In the domain of regenerative medicine research there are many fronts aimed at increasing our understanding of induced pluripotent, embryonic and adult stem cells. Recently a new front has appeared, stem cell niches, the microenvironment that is a crucial component in the regulation of stem cells. New in vitro platforms are continually being created to answer the multitude of biological questions that surround stem cell niches. The transition from in vitro to in vivo is a heavy setback in cellular research due to a huge gap between the two conditions, which can only be reduced if the in vitro conditions better mimic those found in vivo. This project has reduced the gap and has the potential to continue minimizing the differences between the two conditions in the field of muscle stem cell research. Through a combination of the forefront techniques in biological research, we were able to create a new innovative bioengineered platform, a bipolar hydrogel microenvironment. First, we took advantage of polyethylene glycol (PEG) hydrogels, which are highly hydrophilic polymer networks that have properties that mimic physiologic tissue, including high water content and low Young's Modulus. Microfabrication techniques allowed us to topographically pattern the hydrogel at micro-dimensions resembling the native cell environment. Utilizing microfluidics and micro-printing techniques, the three-dimensional hydrogel surface was also patterned with tethered niche proteins found in the in vivo muscle stem cell niche. The swelling properties of the PEG hydrogel were considered as an advantage and controlled to form niche gaps with physical properties that were tuned to correspond to those found in vivo. This new complex and controlled surface for cells to be cultured on is a tool, which will allow biologists to probe new questions and gain insight into the regulatory networks that control cell fate

In vitro endoderm emergence and self-organisation in the absence of extraembryonic tissues and embryonic architecture

Stefano Davide Vianello

In humans, mice, and other mammals key internal organs such as the gut, the lungs, the pancreas, and the liver all derive from the same embryonic tissue: the endoderm. The development of all of these structures thus depends on a same set of early cells, and on the developmental instructions provided to them by the embryo. One approach to study what these instructions might be is to study the behaviour of endoderm cells when they can not rely on embryonic instructions. That is, when they are generated and made to develop outside of the embryo. The rationale behind this approach, widely embraced by the field of synthetic embryology, is that the unguided development of specific embryonic cell types can provide a window to the observation of intrinsic characteristics of these cells and of fundamental developmental principles which might not be executed or manifested within the regulated environment of the embryo. Accordingly, in the work here described I use gastruloids, synthetic models of development made by aggregation of mouse embryonic stem cells in vitro to characterise the developmental behaviour of endoderm cells, in the absence of embryonic cues and in the absence of the extraembryonic tissues that they would usually rely on in vivo. In the first chapter of this thesis, I provide an introduction, overview, and review of our current understanding of the developmental biology of mouse endoderm as this develops in the embryo. I also introduce gastruloids as stem-cell-based experimental systems to study developmental mechanisms in vitro, decoupled from the constraints of experimental work with animals and animal embryos. Chapter 2 is dedicated to the earliest steps of endoderm development in gastruloids. I show when and where these cells appear, and how they sort as the gastruloid starts to mature. I find that endoderm cells follow similar spatial and temporal developmental patterns as those they would follow if they were in the embryo. I notably find that they always remain in an epithelial state, even as other cell types undergo mesenchymal transition. In Chapter 3 I describe surprising dynamics of endoderm cells over time, and how they self-organise into complex architectures at the core of the gastruloids. The epithelial domain that forms is dynamic and plastic, and partitions the gastruloids into interfacing compartments. Importantly, I reveal a significant endodermal component to this in vitro system, usually considered to be unstructured and neural/mesodermal in character. In Chapter 4 I show how the core endoderm compartment that I see forming in gastruloids is patterned along the longitudinal axis of the aggregate, with different cellular identities at different anterior-posterior position of the domain. By comparison with published embryonic datasets, I identify endodermal identities corresponding to the full spectrum of those that make up the gut tube in the embryo. Specifically, I describe how most endodermal cells adopt an early intestinal fate, and how many acquire anterior foregut identities. The work described in this thesis presents gastruloids as promising in vitro systems for the study of endoderm biology, morphogenesis, and mature fate specification, filling a gap within the field of synthetic embryology. It also notably opens translational possibilities to the use of gastruloids to produce endodermal cell types which may be otherwise difficult to generate using conventional directed differentiation methods.

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.
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