The authors wish to acknowledge support from the National Nature Science Foundation of China (82001629, XQS), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116, XQS), and Jiangsu Province Postdoctoral Research Funding (2021K277B, XQS).

All of the animal experiments described were approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School&#39;s Committee on the Use and Care of Animals (No. 20171202). All operations follow appropriate animal care and use agency and national guidelines.
NOTE: Mice were raised in a specific pathogen-free (SPF) environment, with a temperature of 22 &#176;C &#177; 1 &#176;C, relative humidity of 50% &#177; 1%, a light/dark cycle of 12 h/12 h, and free access to food and water.
1. Mouse ovariectomy
<ol>
	<li>Sterilize the surgical instruments with a high-temperature and high-pressure sterilizer 1 day before the operation.</li>
	<li>Weigh 8-week-old C57BL/6 mice and intraperitoneally (i.p)&#160;inject 1% sodium pentobarbital accordingly to anesthetize the mice. The dose used is 40 mg/kg17. This dose provides 40-60 minutes of sedation, which meets the requirements of an ovariectomy. For pre-operative analgesia, inject mice with 5 mg/kg meloxicam (s.c). 
	NOTE: Successful anesthesia in mice can be indicated by the disappearance of deep muscle reflexes and steady breathing. Signs of successful anesthesia include no blinking when touching the inner canthus, no swallowing when pulling the tongue, no leg bending when pinching the skin between the toes, and no bouncing when needling the tail.</li>
	<li>Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia.</li>
	<li>Start the operation when mice are completely anesthetized. Place and fix the mice in the prone position on the operating surface, and shave the hair on the back after wetting them with soapy water. Disinfect the skin with 70% ethanol after shaving.</li>
	<li>Find the operation incision site at 0.5 cm (or 1.0 cm below the rib) on the upper edge of the thigh root of both hind limbs parallel to the spine (Figure 1A). 
	NOTE: A correct incision location is essential for quickly localizing the ovary.</li>
	<li>Take the incision site as the center and disinfect the incision area with iodophor 3x, then place a surgical drape around the incision area.</li>
	<li>Make a longitudinal incision of about 0.5-1.0 cm using scissors. Cut the fascia and passively separate the muscles with tweezers.</li>
	<li>Find a piece of the white fat pad in the incision field close to the lower pole of the kidney through the thin muscle layer (Figure 1B). 
	NOTE: The white fat pad is the most obvious indicator of the location of the ovary.</li>
	<li>Clamp the fat mass with hemostatic clips, and pull the ovary wrapped by the fat pad out of the incision.</li>
	<li>Clamp the junction of the ovary and fallopian tube with curved tweezers, and remove the ovary along the ovarian pedicle with scissors. Stop the bleeding using an electrocoagulation pen or a needle of a 1 mL syringe heated on the alcohol lamp. 
	NOTE: Be sure to preserve the fallopian tube, which is the anatomical landmark for the subsequent injection of sesame oil into the uterine horns.</li>
	<li>Rinse the surgical incision with normal saline to prevent tissue adhesion, use a 4-0 suture to sew the muscle and skin respectively, and disinfect the surgical incision with iodophor again.</li>
	<li>Place the mice in the prone position in the cage and resuscitate them in a 25 &#176;C incubator equipped with a light source and a ventilation system for about 2 h.</li>
	<li>Pay attention to the mice after the operation, put them back in the original feeding place alone until they recover completely, and give them enough water and food.</li>
</ol>
2. Postoperative rest and estrogen and progesterone formulation
<ol>
	<li>Make sure the mice rest for 2 weeks after ovariectomy.</li>
	<li>In an ultra-clean cabinet, add estrogen (2 ng/&#181;L) and progesterone (0.2 ng/&#181;L) into sesame oil in RNA enzyme-free centrifuge tubes.</li>
	<li>Place the centrifuge tubes in a thermostatic water bath at 37 &#176;C and shake the tubes continuously to accelerate dissolution.</li>
	<li>After complete dissolution, divide the sesame oil solution into 100 &#181;L aliqouts for convenient use. Store the prepared estrogen and progesterone solutions in a refrigerator at 4 &#176;C.</li>
</ol>
3. Induced artificial decidualization model
<ol>
	<li>Subcutaneously (s.c) inject 100 ng of estrogen&#160;in 50 &#181;L of sesame oil into each mouse for 3 days, followed by 2 days of rest15. 
	NOTE: Ensure that the estrogen and progesterone have been formulated and placed in different places. Any contamination of estrogen by progesterone&#160;may lead to failure.</li>
	<li>Inject (s.c.) 1 mg of progesterone and 10 ng of estrogen&#160;in 50 &#181;L of sesame oil into each mouse for another 3 days15.</li>
	<li>Operate the mice to induce the artificial decidualization model15 6 h after the third estrogen-progesterone combined injection.</li>
	<li>Find the uterus horn at the lower end of the fallopian tubes and inject 20 &#181;L of sesame oil slowly along with the uterine horn, push the tissue back, suture the incision, and allow the mouse to recover. The approach to operation is the same as the mouse ovariectomy15.&#160; 
	NOTE: Only proceed with the second surgery in the event of a successful ovariectomy (skin incision is well healed, the ovary is completely excised, and the residual end of the fallopian tube is not adhered to the surrounding tissues). 
	NOTE: For inducing the AD model, 20 &#181;L of sesame oil is appropriate; more than 20 &#181;L of sesame oil will easily penetrate the other side.</li>
	<li>Inject (s.c.) 50 &#181;L of sesame oil containing 1 mg of progesterone and 10 ng of estrogen (H.) for another 4 days. See details in Figure 215,18.</li>
</ol>
4. Sample collection
<ol>
	<li>Euthanize the mice (n = 6) by carbon dioxide asphyxiation and collect the uteri. Observe the shape of the bilateral uteri, take photos, and weigh the uterine horns (Figure 3A, B).</li>
	<li>Fix 3-5 mm of the uterus tissue with 10% formalin, embed in paraffin, and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes19 (Figure 4A).</li>
	<li>Embed another 3-5 mm of the uterus tissue in optimal cutting temperature compound (OCT), freeze in liquid nitrogen, and store in a &#8722;80 &#176;C refrigerator. Frozen section the tissue to 10 &#181;m and detect alkaline phosphatase activity in the uterine tissue by the azo coupling method20 (Figure 4B). 
	NOTE: Satisfactory results can be obtained using frozen sections to detect ALP content, not paraffin sections.</li>
	<li>Extract the total RNAs of the uterus with Trizol reagent and perform quantitative real-time PCR (qPCR) on a real-time PCR system (Table of Materials) with qPCR master mix (Table of Materials) to detect the relative mRNA expression of prolactin family 8 subfamily member 2 (Prl8a2), alkaline phosphatase liver/bone/kidney (Alpl), and insulin-like growth factor-binding protein 1 (Igfbp1)15 by normalization to 18S mRNA levels (Figure 4C-E). Follow the PCR conditions: Stage 1, 95 &#176;C for 30 s; Stage 2, 95 &#176;C for 10 s and 60 &#176;C for 30 s; repeat the cycle 40x. A complete list of primer sequences is provided in Table 1.</li>
	<li>Analyze qPCR data using the 2-&#916;&#916;CT method and a t-test method for statistical analysis in the appropriate software. In this study, p &#60; 0.05 was considered statistically significant.</li>
</ol>

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The authors have nothing to disclose.

Decidualization in mice is a spontaneous process depending on the presence of embryos, which is different from humans. However, it has been found that artificial stimulation such as uterine injection of glass beads and uterine laceration can induce decidualization of the endometrium instead of embryos. In addition, researchers found that many factors could induce decidual or participate in decidualization, such as the injection of steroid hormones, prostaglandins, and growth inhibitory factors into the uterine cavity<sup...

With the development of human-assisted reproductive technology, the current clinical pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) has reached or even exceeded that of natural pregnancy. Despite this, many patients in assisted reproduction clinical practice still undergo multiple embryo transfers but fail to achieve pregnancy as desired. However, its specific molecular mechanism is still unclear, so clinical intervention is ineffective, which is one of the significant challenges facing reproductive medicine1,2.
Endometrial factors account for about two-thirds of the causes of IVF failure3. Human embryo implantation is divided into three stages: positioning, adhesion, and invasion4,5,6. The maternal endometrium undergoes a series of changes to meet the arrival of the embryo. Forming an implantation &#34;window period&#34; provides favorable conditions for embryo implantation7,8.
In most mammals, after the blastocyst adheres to the luminal epithelium of the uterus, the stromal cells surrounding the blastocyst rapidly begin to proliferate and differentiate, and the rapid remodeling of the mesenchyme changes its shape and function, leading to embryo implantation5,9,10. The rapid increase in site volume and weight allows the blastocyst to become embedded in the uterine stroma, a process known as decidualization11. The endometrial stroma differentiates and remodels in preparation for pregnancy, while the transition of stromal cells provides space and new signaling connections for decidual cells to perform their functions12,13. Stromal cells transform into decidual cells and secrete many iconic factors such as prolactin (PRL), insulin-like growth factor-binding protein 1 (Igfbp1), and so on. Studies have shown that abnormal decidualization is one of the key reasons for embryo implantation failure, but the cause of abnormal decidualization is still unclear and needs to be further elucidated1,14.
The mouse artificial decidualization model is essential for studying the physiological process and molecular mechanisms underlying decidualization. Artificial decidualization (AD) mainly refers to the process of endometrial decidualization established by artificial methods to simulate pregnancy or the menstrual cycle. In terms of morphology, there is little overall difference between pregnancy decidualization and artificial decidualization15,16. The uterine glands exist in the endometrium before decidua form and disappear after decidualization. Regarding the gene expression, only a slight difference is identified between natural pregnancy decidualization (NPD) and AD15. Consequently, the artificial decidualization model in mice can simulate pregnancy decidualization to explore the unknown pathogenesis and new treatment of human reproductive diseases.
NPD, AD, and in vitro decidualization (IVD) are three methods to achieve mouse decidualization. The NPD model depends on natural pregnancy and is closest to the maternal physiological state, including the effects from embryos. Comparing the differences between implantation and non-implantation sites is a more physiological and convenient approach for studying decidualization. The AD model was developed by using an intrauterine injection of sesame oil as a stimulant to induce decidualization in a pseudopregnant female mouse mated with vasectomized males to avoid the impact from embryos. Both NPD and AD models play essential roles in different research purposes, but they cannot avoid mating failure and within-group differences caused by the different activities of maternal hormone metabolism. IVD is a method depending on the treatment of combined estrogen and progesterone at the cellular level, which requires more stringent experimental conditions and operating ability. However, the in vitro model cannot fully simulate the decidual response under physiological conditions15. Therefore, we propose a simple and improved induction method modified from traditional AD to reduce the effect of endogenous hormones on decidualization. Based on ensuring the success of decidualization induction, it is closer to the physiological state and more suitable for experiments that need to exclude embryo factors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Estrogen</td>
			<td >Sigma</td>
			<td >E2758</td>
			<td >Hormone supplement</td>
		</tr>
		<tr >
			<td >Progesterone</td>
			<td >Sigma</td>
			<td >P0130</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sesame oil&#160;</td>
			<td >Sigma</td>
			<td>S3547</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sodium pentobarbital&#160;</td>
			<td >Dainippon Sumitomo Pharma Co.,Ltd.</td>
			<td ></td>
			<td>Anaesthesia</td>
		</tr>
		<tr >
			<td >Meloxicam injection</td>
			<td >Qilu Animal Health Products Co., Ltd</td>
			<td ></td>
			<td>Analgesia</td>
		</tr>
		<tr >
			<td >Alkaline phophatase stain kit(kaplow&#39;s/azo coupling method)</td>
			<td >Solarbio</td>
			<td>G1480</td>
			<td>Alkaline phophatase stain</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td >Servicebio</td>
			<td>G1005-2</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td >Servicebio</td>
			<td >G1005-1</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >ChamQ Universal SYBR qPCR Master Mix</td>
			<td >Vazyme</td>
			<td>Q711-02</td>
			<td>qPCR</td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >Lircon</td>
			<td>ZH1120090</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Iodophor</td>
			<td >Runzekang</td>
			<td >RZK-DF</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Erythromycin Eye Ointment</td>
			<td >Guangzhou Baiyunshan</td>
			<td ></td>
			<td>Mice eyeball protect</td>
		</tr>
		<tr >
			<td >4-0 suture</td>
			<td >Ethicon</td>
			<td >W329</td>
			<td>Incision suture</td>
		</tr>
		<tr >
			<td >10% formalin</td>
			<td >Yulu</td>
			<td >L25010118</td>
			<td>Tissue fix</td>
		</tr>
		<tr >
			<td >Optimal cutting temperature compound</td>
			<td >Sakura</td>
			<td >4583</td>
			<td>Ssection</td>
		</tr>
		<tr >
			<td >Trizol reagent</td>
			<td >Ambion</td>
			<td >15596018</td>
			<td>qPCR</td>
		</tr>
	</tbody>
</table>

generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

The mouse decidualization model indexes include the uterus&#39;s general morphology, the mass ratio of the decidualized and non-decidualized uterus, the histological morphology of the endometrium, and the expression level of decidualization marker molecules. The general morphology of the artificial decidualized uterus of mice induced by oil is closer to that of the uterus in pregnancy. The uterine body becomes thick, and the uterine cavity becomes smaller than the non-induced side. The volume and weight of the induced ut...

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Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

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4.1K Views.  The Affiliated Drum Tower Hospital of Nanjing University Medical School. Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Video: Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

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 ...

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 ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...