This work was supported in part by grants from the Hainan Provincial Natural Science Foundation of China (824MS152).

We have complied with all relevant ethical regulations for animal testing and research. All experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the Shanghai Ninth People&#39;s Hospital, School of Medicine, Shanghai Jiaotong University.
Both the Rosa26-loxp-stop-loxp-dnRAR&#945;403 strain (R26dn/dn) and the Osterix-Cre (OsxCre) (No.006361) strain of mice used here were maintained on the C57BL/6 background. R26dn/dn mice were crossed with OsxCre mice to generate OSXCre;R26dn/dn mice. All mice were bred and maintained under specific pathogen-free (SPF) conditions.
1. Breeding of mice
NOTE: F means the number of generations of mice; N means the number of generations of mating between zygotic mice and background mice. Thus, F2+N represents the second generation as well as any subsequent mouse breeding programs. F0 means primary mice.
<ol>
	<li>Pair a sexually mature male mouse with a pair of female mice of a comparable age. Commence daily inspections for newborn pups after an initial period of 18 days. Upon identifying pregnant females, isolate them if necessary to ensure optimal maternal care. In the event that no pregnancies are observed within a month of the initial pairing, consider alternating the male mouse among different breeding setups to promote fertilization.</li>
	<li>Cross R26dn/dnmice withOSXCre mice (F0). Clip the tails for genotyping and keep the male OSXCre;R26dn/+mice in cages until sexually mature, which is ~6 weeks of age (F1).</li>
	<li>Cross six-week-old male OSXCre;R26dn/+mice with female R26dn/dnmice (F2). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F2+N).</li>
</ol>
2. Preparation
<ol>
	<li>Prepare four pairs of 4-week-old OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. Individually euthanize the mice through the administration of carbon dioxide asphyxiation. 
	NOTE: To ensure efficient euthanasia, the CO2 flow rate should be adjusted to displace approximately 30% of the cage&#39;s volume every minute. For instance, in a cage measuring 45 cm x 30 cm x 30 cm, a flow rate of 40 L/min is recommended.</li>
	<li>Cut the neck of the mouse with scissors while holding its body, leaving the skull intact. 
	NOTE: To obtain a complete skull, open the mouth of the mouse, cut along the corner of the mouth with ophthalmic scissors, pull both hands to both sides, and rotate to peel the skin of the mouse&#39;s head.</li>
	<li>Immerse the mouse skulls in 4% paraformaldehyde for 48 h, then store them in 70% ethanol. 
	CAUTION: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>Scan the collected skulls with a microCT scanner- resolution: 4.5 &#181;m; voltage: 70 kV; current: 114 &#181;A; filter: 0.5 mm Al; rotation step: 0.5&#176;.</li>
	<li>Store CT scan images. Images in DICOM format are selected for import into the software and analyzed.</li>
</ol>
3. MicroCT imaging and 3D reconstruction
NOTE: All bones utilized in this study were manually segmented.
<ol>
	<li>Click the right mouse button on the MASKs page and select New mask; a new layer named Green appears.</li>
	<li>Select the threshold range of 671-2,566 in the Thresholding page that pops up. Repeat this process for all related layers. (Figure 1A).</li>
	<li>Use the Eraser tool in the Edit menu to manually erase the bones attached to the mandible in each layer on the 2D sagittal plane (Figure 1B).</li>
	<li>Select the region of the mandible on the 2D sagittal plane after clicking on the Region Growing option (Figure 1C). 
	CAUTION: Ensure that the mandibular region is selected completely in each layer; otherwise, it will result in an incomplete mandible after reconstruction.</li>
	<li>Click on the newly created layer Mandible in the MASKS page and select Calculate 3D from the dropdown menu (Figure 1D).</li>
	<li>In the 3D objects interface, right-click Mandible and click Properties. Click to change Color. Color code all the 3D models reconstructed with different bones.</li>
	<li>Click on Measure in the upper left corner and select distance from the dropdown menu that appears. Calculate the length by marking points in the 3D image (Figure 1E). 
	CAUTION: The reference for measuring bone width is the widest part of the bone.</li>
	<li>In the 3D-objects interface, right-click Mandible and click Properties. Properties-Info-Volume module to read the reconstructed volume values for each craniomaxillofacial bone block. (Figure 1F).</li>
	<li>Measure the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone in the same way as above and mark them individually with different colors.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Perform statistical analysis using the software of choice. Perform two-tailed Student&#39;s t-tests (n = 4/group).</li>
	<li>For all graphs, use error bars to represent standard deviations. Consider P-values &#60; 0.05 statistically significant.</li>
</ol>

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The authors have no conflicts of interest to disclose.

MicroCT is a powerful tool for obtaining realistic and isotropic 3D information from dense and opaque biological samples with micrometer resolution. The data obtained from microCT are calibrated for geometry and intensity, making it especially useful for quantitative studies13,14,15,16,17. It is used to study bone and dental microstructure18<...

The intricate development of the human skull and face encompasses a sophisticated 3D morphogenetic process, intricately orchestrated by numerous genes. These genes play a pivotal role in regulating the intricate patterns, proliferation, and differentiation of tissues derived from diverse embryonic sources. This highly coordinated process underscores the complexity of human craniofacial growth and development. Craniofacial malformations (including cleft lip and palate, cranial suture closure, and facial hypoplasia) occurring as a result of developmental abnormalities account for more than one-third of all congenital birth defects. As a commonly used model animal in biomedical research, the mouse has a complex and delicate craniomaxillofacial bone structure that is very similar to the human craniomaxillofacial bone in terms of anatomy and physiology. The study of craniomaxillofacial developmental biology has come a long way in recent years with the advent of new techniques in mouse genetics, especially in malformations1.
Retinoic acid (RA) is the in vivo metabolite of vitamin A2. Vitamin A deficiency (VAD) is associated with a range of serious multisystem disorders, such as poor bone remodeling, fractures, as well as craniofacial malformations and skeletal malformations characterized by dwarfism3,4 . Retinoid receptors (RARs) are crucial transcription factors in retinoid signaling5. A dominant-negative RAR&#945;403 mutant (dnRAR&#945;) was designed6 and a mouse model established in which osteoblasts expressed dnRAR&#945;. This resulted in the mice exhibiting dwarfism, craniofacial deformities, incomplete cortical bone formation, and increased but poorly remodeled trabecular bone.
Microcomputed tomography (microCT) has great potential for the study of craniomaxillofacial malformations. It possesses the capability to detect and track the evolution of both innate and acquired skeletal abnormalities in rodent models. MicroCT imaging analysis offers an in-depth exploration of craniofacial growth disturbances in genetically modified mouse models7,8 .Furthermore, 3D imaging emerges as a vital tool for delineating morphological traits, facilitating tailored analysis and visualization approaches9. Micro-CT has been used in several studies to analyze craniofacial phenotypes, including defining anatomical landmarks in humans and mice and volumetric analysis of each craniofacial bone10,11,12. Here, we describe in detail a method based on microCT technology to separate and measure 3D models of mouse craniomaxillofacial bones to enable better evaluation and analysis of mouse craniomaxillofacial skeletal development than is possible with current methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GraphPad Prism 6.01 Software</td>
			<td>GraphPad Software Inc., La Jolla, CA, USA</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Quantum GX micro CT, PerkinElmer, 
			Waltham, MA, USA</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Mimics Medical 19.0&#160;</td>
			<td>Materialise, Leuven, Belgium</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Osterix-Cre (OsxCre)&#160;</td>
			<td>/</td>
			<td>/</td>
			<td>from the Jackson Laboratory</td>
		</tr>
		<tr>
			<td>Rosa26-loxp-stop-loxp-dnRAR&#945;403 strain</td>
			<td>/</td>
			<td>/</td>
			<td>from the Columbia University, USA</td>
		</tr>
	</tbody>
</table>

analysis of craniomaxillofacial malformations in mice using three-dimensional microcomputed tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Extensive research underscores the multifaceted impact of genetic mutations on mouse growth, development, and organ systems. A comprehensive evaluation of craniofacial bones in mutant mice necessitates methods beyond single-tissue or 2D image analysis due to their limitations. Therefore, elucidating craniofacial bone development holds paramount importance for investigating human craniofacial disorders.
This method provides a use...

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

analysis-craniomaxillofacial-malformations-mice-using-three

Research

JoVE Journal

Developmental Biology

128 Views.  Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Video: Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...