This work was supported by NIGMS (R01GM11755) to P.W.J. and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to S.R.W. and J.H. 
&#160;

The use of mice was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University. Experiments were performed on juvenile (20 - 26 days post-partum, dpp) C57BL/6J mice, taking advantage of the semi-synchronous first wave of spermatogenesis. However, this technique can also be performed using adult mice.
1. Dissection and isolation of mouse seminiferous tubules
<ol>
	<li>Prepare the fix/lysis solution and antibody dilution buffer (ADB) described in Table 1 and Table 2.</li>
	<li>Prepare one 35-mm Petri dish with 3 mL of 1x phosphate buffered saline (1x PBS, pH 7.4), and another 35-mm dish with 2 mL of fix/lysis solution per mouse.</li>
	<li>Sacrifice the mouse via cervical dislocation or CO2 asphyxiation and spray the ventral abdomen with 70% ethanol. Open the abdominopelvic cavity using sterile scissors, making a V-shaped opening. Then remove the testes by pulling on the epididymal fat pad using forceps, and avoid disturbing the tunica albuginea. Place the testes in a 35-mm Petri dish containing 1x PBS. 
	NOTE: Utilize the first wave of spermatogenesis in order to observe an enriched cell population of interest14. Specific stages of spermatogenesis are enriched at different mouse ages (Table 3).</li>
	<li>Remove the testicular tunica albuginea by puncturing the tissue with sharp tipped forceps and collect the loose seminiferous tubules. Transfer the tubules to a new 35-mm Petri dish containing 2 mL of fix/lysis solution.</li>
	<li>Incubate the tubules in the fix/lysis solution for 5 min at room temperature.</li>
</ol>
2. Preparation of seminiferous tubules
<ol>
	<li>Prepare a poly-L-lysine coated glass slide by outlining the edges of the slide with a liquid blocker pen.</li>
	<li>Apply 100 &#181;L of fix/lysis solution to the center of the glass slide.</li>
	<li>Using sterile forceps and scissors gently tease apart the seminiferous tubules. Cutting out long individual tubule segments approximately 20 mm in length. 
	NOTE: To visualize structures that are sensitive to prolonged fixative exposure, such as the centrosomes, Separate the tubules first in the 1x PBS, then directly mince the tubules on the surface of the poly-lysine slides coated in 100 &#181;L of fix-lysis solution.</li>
	<li>Transfer five 20 mm long seminiferous tubule segments to the prepared poly-L-lysine coated glass slide containing fix/lysis solution.</li>
	<li>Using sterile scissors mince seminiferous tubules into 1.5 to 3.0 mm segments.</li>
	<li>Using sterile forceps arrange the tubule segments on the glass slide so that no tissue overlaps, and the tubules are distributed evenly. 
	NOTE: The optimal number of tubule segments on a glass slide is between 20 and 40.</li>
</ol>
3. Squashing of the seminiferous tubules
<ol>
	<li>Transfer the glass slide containing dispersed seminiferous tubule segments onto a benchtop. Remove excess liquid using a laboratory wipe to avoid losing tissue during the squash step.</li>
	<li>Apply a coverslip (22 x 60 - 1.5 mm) on top of the glass slide and apply pressure with the heel of the palm for 10 - 20 seconds. It is important to apply enough force to disperse spermatocytes from the seminiferous tubules. 
	NOTE: Observe the tubules using a dissection microscope in order to optimize the squashing step so that all tubule segments are disrupted.</li>
	<li>Using slide forceps, immediately flash freeze the glass slide in a small dewar of liquid N2 for 15 seconds, or until the liquid ceases to bubble. If immunolabeling immediately, remove the coverslip with a straight edge razor blade, fine tip forceps or 21-gauge needle. 
	NOTE: For optimal results, immunolabel the slides immediately (see step 4). However, at this point slides can be preserved at -80 &#176;C for up to two weeks. To maximize the lifetime of proteins and cellular structures, immediately store the slides on dry ice or transfer to a -80 &#176;C freezer, and keep the coverslip on the slide. When immunolabeling stored slides, remove the coverslip by immersing the slides in liquid N2 for 15 seconds, as described in step 3.3.</li>
</ol>
4. Immunolabeling of mounted seminiferous tubules
<ol>
	<li>Immerse the slide in 1x PBS, and wash three times for 5 min in a 50-mL Coplin jar containing 1x PBS. 
	NOTE: Never allow the slides to completely dry during immunolabeling.</li>
	<li>Apply 1 mL of antibody dilution buffer onto the glass slide to block for 1 - 2 h in a humidified chamber.</li>
	<li>Remove the antibody dilution buffer and apply 100 &#181;L of primary antibody diluted in antibody dilution buffer in a humidified chamber. 
	NOTE: For best results, incubate primary antibodies overnight at 4 &#176;C. Use smaller volumes of antibody (e.g. 50 &#181;L) by covering the slide with a coverslip or parafilm. Validate and optimize primary antibodies15.</li>
	<li>Rinse slides three times for 5 min in a 50 mL Coplin jar containing 1x PBS. 
	NOTE: To enhance washes, agitate Coplin jars by using a small magnetic stir bar and place on a magnetic stir plate at low speed, or place the Coplin jar on a tabletop mixer at low speed.</li>
	<li>Apply 100 &#181;L of secondary antibody diluted in antibody dilution buffer for 1 to 1.5 h in a humidified chamber at room temperature. Incubate the slides in a dark box to avoid photo bleaching of fluorophore conjugated antibodies. 
	NOTE: Use smaller volumes of antibody (e.g. 50 &#181;L) by covering the slide with a coverslip or parafilm.</li>
	<li>Rinse slides two times for 5 min in a 50-mL Coplin jar containing 1x PBS.</li>
	<li>Mount the slides in mounting medium containing 4&#39;,6-diamidino-2-phenylindole (DAPI, 1.5 &#181;g/mL) and apply coverslips (22 x 60 -&#160;1.5 mm).</li>
	<li>Seal the coverslips to the slides with clear nail polish to preserve the slides and prevent the coverslips from moving.</li>
</ol>
5. Analysis and imaging tubule squash preparations
<ol>
	<li>Use an epifluorescence microscope with automated stage that enables precise Z-axis movement and a high-resolution camera for image capturing. Alternatively, use a laser confocal microscope. 
	NOTE: Use any available hardware and software for this step. For example, images displayed in Figure 1 and Figure 2 were captured using a Zeiss Cell Observer Z1 linked to an ORCA-Flash 4.0 CMOS camera and analyzed with the Zeiss ZEN 2012 blue edition image software.</li>
	<li>Assess the slides using a 20X&#160;objective to determine the quality of the squash preparation. Good quality slides have a monolayer of nuclei that are evenly distributed. 
	NOTE: Some sections of the slide may be better than others, it is useful to note the coordinates of where the best regions of the slide can be found for further analysis using higher magnification.</li>
	<li>Using higher magnification objective (e.g. 63X&#160;or 100X), capture regions of the slide that have a consistent monolayer of nuclei. Once a region is selected, set the upper and lower Z-stacks to ensure that all in-focus light is captured. 
	NOTE: The optimal number of Z-stacks captured between the upper and lower limit is generally designated by the image acquisition software and is different for each objective.</li>
	<li>Use the image processing software to compile an extended depth focus image. The software combines the in-focus light from each Z-stack, and is essential for optimal imaging of tubule squash preparations.</li>
</ol>

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The authors have nothing to disclose.

Mice have proven to be a useful model organism for studying the cellular events that govern meiotic progression during spermatogenesis. Further, it is necessary to develop tools catered to the study of spermatogenesis because many events, such as exit from meiotic prophase I, are sexually dimorphic. This protocol describes a seminiferous tubule squash method for visualization and study of different stages of mouse spermatogenesis. This method preserves cellular integrity and thereby allows detailed analyses of nuclear an...

Meiosis is a complex cellular event in which a single round of DNA replication is followed by two successive rounds of cell division. Several meiosis-specific events must be coordinated during the initial stages of meiosis to ensure accurate chromosome segregation. These events include the completion of homologous recombination, co-orientation of sister kinetochores during the first meiotic division, and the stepwise loss of cohesin complexes to resolve chiasmata between homologs. Precise regulation of these processes is necessary to maintain fertility and to prevent chromosome missegregation events that can lead to genetic developmental disorders and spontaneous miscarriage1.
While the key events of meiosis take place in both males and females, significant temporal and mechanistic differences exist between spermatogenesis and oogenesis2. For example, during female meiosis, prophase I occurs during embryonic development and arrests at the dictyate stage until puberty. In contrast, spermatogenesis commences at puberty and progresses in waves throughout adult life without arrest. The differences between male and female meiosis emphasizes the need to develop methods that are specifically catered towards assessing these processes in both spermatocytes and oocytes. Currently, assessing meiotic progression largely relies on the use of chromatin spreads3,4,5. While chromatin spreads are useful for studying meiotic chromosomes, they fail to preserve cellular integrity, preventing evaluation of cellular structures such as spindle microtubules, centrosomes, the nuclear envelope, and telomere attachments. Live imaging and long-term culturing techniques have greatly advanced our understanding of female meiosis; similar approaches to visualize the entire intact cell, however, are less frequently implemented for the study of spermatogenesis6,7. In order to visualize dynamic events throughout male meiosis, we have adapted established tubule squash techniques to rapidly assess the cytological features of developing mouse spermatocytes8,9. The method described here maintains the integrity of the cell, enabling the study of multiple cellular structures during different stages of spermatogenesis.
This tubule squash technique is a whole cell approach, which allows for the assessment of cellular structures via immunofluorescence microscopy. Common histological approaches to visualize meiotic progression in male mice such as haematoxylin and eosin staining of paraffin embedded testes, and immunofluorescent labeling of cryosections allow for a broad overview of meiotic progression. However, these techniques fail to resolve single cells to the extent necessary for detailed analysis of the events occurring throughout meiosis10,11. Alternative techniques to visualize meiotic processes rely on significant chemiosmotic disruption to the spermatocyte to isolate and fix nuclear materials3,4,5. These chemical treatments hinder the observation of cell types other than primary spermatocytes. A recently described method by Namekawa has enabled the research community to preserve the nuclear architecture of isolated spermatocytes, but requires the use of a cytospin and accessories that may not be readily available to some laboratories4. In contrast, the tubule squash technique only requires equipment that is generally standard in most cell biology laboratories.
The tubule squash method described here can be used to visualize the diverse cell types found within the seminiferous tubule, including sertoli cells, spermatogonia, primary and secondary spermatocytes, and spermatids. By coupling this technique with the near-synchronous first wave of spermatogenesis in juvenile mice, it is possible to obtain enriched populations of spermatogenic cells as they progress through meiosis12. This process permits the detailed analysis of processes throughout spermatogenesis, such as early prophase events, the G2/MI and metaphase to anaphase transitions, and spermiogenesis. Furthermore, tubule squash preparations can be used to visualize cytological features of the chromosomes (e.g. interchromatid domains (ICDs) and kinetochores) and centrosomes (centrioles and pericentriolar material/matrices). The squash method can be readily performed in parallel with other experimental approaches, such as chromatin spreads and protein extraction. In addition, this technique has been successfully modified to deposit living spermatogenic cells on slides for direct visualization13.
The method described here involves a whole cell seminiferous tubule squash technique to analyze the G2/MI transition in wild-type C57BL/6J mice. The cytological features of primary spermatocytes entering the first meiotic division were visualized with immunofluorescence microscopy to observe the meiotic spindle. This versatile technique can be easily modified to visualize other meiotic stages and different cell types. The technique is also amenable to alternative visualization strategies, such as DNA and RNA FISH approaches.

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			<td style="width:167px;">1 in 50</td>
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			<td height="21" style="height:21px;width:251px;">Rabbit anti-SYCP3</td>
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			<td height="21" style="height:21px;width:251px;">Mouse anti-AIM1</td>
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			<td height="21" style="height:21px;width:251px;">Mouse anti-&#947;H2AX</td>
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			<td height="21" style="height:21px;width:251px;">Mouse anti-CENT3</td>
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			<td height="21" style="height:21px;width:251px;">Rabbit anti-REC8</td>
			<td style="width:247px;">Courtesy of&#160; Dr. Karen Schindler</td>
			<td style="width:137px;">N/A</td>
			<td style="width:167px;">1 in 1000</td>
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a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

Here, we have used the tubule squash method to visualize transitory cell populations undergoing the prophase to metaphase I (G2/MI) transition, which were enriched by harvesting testes from juvenile wild-type mice undergoing the first wave of spermatogenesis (24 dpp). Figure 1 depicts representative images of the various cell stages that can be visualized using the tubule squash method. Enriched populations of metaphase I spermatocytes were visualized using a...

Watch this Scientific Journal Video about A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model at JoVE.com

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

a-seminiferous-tubule-squash-technique-for-cytological-analysis

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Developmental Biology

14.8K Views.  Johns Hopkins Bloomberg School of Public Health. The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Video: A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

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 ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.