Name: preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy
Uploaded: 2017-05-18T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

This research was supported by the Li Ka Shing Foundation, the Guangdong High-Level University Project &#34;Green Technologies for Marine Industries,&#34; the Natural Science Foundation of China (http://www.nsfc.gov.cn/ Grant No. 30971665, 81172894, 81370925), and the Education Department of Guangdong (http://www.gdhed.edu.cn/ Grant No.cxzd1123).

8 to 12 week-old BALB/c mice were purchased from Shanghai Experimental Animal Center (Shanghai, China) and raised in a specific pathogen-free and air-conditioned animal facility. Animal care and experimental procedures were in compliance with the guidelines for the use and care of laboratory animals established by Shantou University.
1. Assembly of the Apparatus
<ol>
	<li>To ensure sterility, turn on the UV light of a tissue culture hood for 30 min before use.</li>
	<li>Unscrew a disposable ...

<ol>
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Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

A tremendous amount of effort has been devoted to establishing approaches for gene, enzyme, and cell replacement therapies. This has resulted in great breakthroughs and even clinical applications1,2,3. Recently, a controversial mitochondria replacement therapy based on nucleus transfer technology was applied to in vitro fertilization for women of old age or carrying a lethal mitochondrial DNA mutation4. Defects found in age-related diseases, including atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer, are usually multi-faceted. It has been documented that the accumulation of lipid droplets; the deposition of amyloid protein; the retention of unfolded proteins in the endoplasmic reticulum; and defective proteasome, autophagosome, and mitochondria contribute to the development or aggravation of these diseases5,6,7,8,9,10,11. Presently, there is no available mechanism aimed at direct remediation of malfunction in the cytosol and organelles, which causes senescence and ageing phenotypes.
We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells12. With the exception of the nucleus, components in the membrane or cytosol, including proteins and RNA, as well as the organelles, such as mitochondria, were found in PMVs. Essentially, a PMV can be regarded as a miniature enucleated cell. More importantly, the fusion of PMVs with mitochondria-deficient Rho0 cells restored mitotic activity under normal culture conditions. This is the first report on establishing a potentially efficient approach for cytoplasm replacement therapy.
Bone marrow mesenchymal stem cells (BMSCs) are multipotent progenitor cells that are routinely generated from the bone marrow and are readily expanded in culture. Embryonic stem cell markers Oct4, Nanog, and SOX2 have been detected at low levels in MSCs13. Telomerase activity is also measurable. In addition, the absence of co-stimulatory molecules and human leukocyte antigen (HLA) Class II molecules, as well as low HLA Class I expression on MSCs, make them ideal cells for allogeneic, or &#34;off-the-shelf,&#34; use in both regenerative medicine and immunomodulatory applications14.
Here, we describe how to prepare PMVs from mouse BMSCs via extrusion through a polycarbonate membrane with 3-&#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential in PMVs using confocal microscopy, prepare concentrated but not aggregated PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IsoporeTM membranes</td>
			<td>Millipore</td>
			<td>TSTP04700</td>
			<td>3 mm pore</td>
		</tr>
		<tr>
			<td>Disposable filter unit</td>
			<td>Xinya, Shanghai, China</td>
			<td>25 mm</td>
			<td>Medical grade polypropylene</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328446</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>pN1-EGFP</td>
			<td>Clontech</td>
			<td>&#160;6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
			<td>Green FM, 1 &#956;M</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Beyotime, Haimen, China</td>
			<td>C2006</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Beyotime, Haimen, China</td>
			<td>C1036</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td>Mn = 75000</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE 2000</td>
			<td>With CCD camera</td>
		</tr>
		<tr>
			<td>Confocol Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 510 Meta</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>SigaGen</td>
			<td>SL100688</td>
			<td>For cell transfection</td>
		</tr>
	</tbody>
</table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

The key to a successful preparation of PMVs depends heavily on the correct assembly of the filter unit (Figure 1), which can be tested by pushing 1 mL of PBS through the membrane. If leakage occurs, reassemble the filter unit and test again. However, the leakage can only be tested reliably when cells are pushed through the membrane. If only a few PMVs are detected under a regular microscope using the 10X objective, or if the size of the PMVs is mostly around 1 &#181;m, th...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

The overall goal of this procedure is to deliver a novel cytoplasm replacement therapy to the gastrocnemius muscle of mice using plasma membrane vesicles, or PMVs derived from mouse bone marrow mesenchymal stem cells or BMSCs. This method can help answer questions with supplementation of cells with young cytoplasm from other BMSC via PMV fusion can ameliorate or even reverse the age associative phenotypes. The main advantage of this technique is that PMV encapsulation of BMSC cytosol and can be instantly induced by a mechanical extrusions.When you are demonstrating this method it is critical as you're extruding and constructing steps are difficult to learn by testing to actually owning. To generate the PMVs, first culture mouse BMSCs in complete DMEM culture medium in one well of a six well plate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. When the cells have reached 90%confluency, rinse the well with 0.5 milliliters of calcium free PBS and detach the cells with 0.5 milliliters of 0.25%trypsin-EDTA.After three minutes at 37 degrees Celsius, tap the plate with the palm of the hand a few times to facilitate cell dissociation and stop the digestion with one milliliter of culture medium. Transfer the cells into a five milliliter conical tube for centrifugation and resuspend the pellet in four milliliters of fresh culture medium. Then divide the cells between two wells of a new six well plate and return the plate to the cell culture incubator for 24 hours.The next day, harvest the cells from one well by trypsin digestion as just demonstrated. Resuspending the pellet in 0.5 milliliters of fresh culture medium after collection by centrifugation. Load the cells into a one milliliter insulin syringe and attach the syringe to a filter unit.Quickly depress the plunger to squeeze the cells through the filter, and discard the extruded medium. Then load and dispense 0.5 milliliters of extra culture medium through the filter as just demonstrated, collecting the medium from the second extrusion. Seed 150 microliters of the collected medium into a 35 milliliter glass bottom dish and allow the vesicles to settle for about 10 minutes.Then confirm the presence of PMVs under an inverted phase contrast microscope equipped with a CCD camera using a 20 times objective lens. To trace the mitochondrial enclosure and membrane potential in the PMVs, stain a 90%confluent BMSC culture with an appropriate mitochondrial dye or a mitochondrial membrane potential probe respectively in fresh culture medium for 30 minutes at 37 degrees Celsius. Then rinse the cells three times with 0.5 milliliters of PBS per wash and harvest the cells by trypsin digestion for PMV generation as just demonstrated for their visualization by confocal microscopy.To trace the cytoplasmic localization of the PMV proteins, first dilute two micrograms of supercoiled plasmid DNA for an EGFP expression cassette and six microliters of cationic transfection reagent in individual 0.5 milliliter tubes containing 50 microliters of serum free DMEM. Vortex the tubes, followed by a brief centrifugation. Then add the transfection reagent drop wise into the tube containing the diluted DNA solution.Vortex the DNA transfection mixture robustly for one minute, followed by a brief spin in a 10 minute incubation at room temperature. Next, feed a 30%confluent BMSC culture in a six well plate with two milliliters of fresh culture medium and add the DNA mixture drop wise to the cells. 48 hours after their transfection, harvest the BMSC by trypsin digestion and prepare PMVs as just demonstrated.The PMVs can then be analyzed by confocal microscopy using a 100 times oil objective. Before injecting the PMVs, label a BMSC culture with a membrane staining fluorescent cell tracker dye in one milliliter of fresh culture medium at 37 degrees Celsius for 30 minutes and harvest the cells by trypsin digestion. Resuspend the labeled cells in 0.5 milliliters of fresh culture medium supplemented with one microgram of polyethylene amine and prepare PMVs as demonstrated.Then concentrate the PMVs by centrifugation and resuspend the pellet in 100 microliters of fresh culture medium with gentle swirling. Next, load the cells into a one milliliter insulin syringe equipped with a 30 gauge needle and disinfect the outside of the gastrocnemius muscle of an anesthetized BALB/c mouse with 75%ethanol. Then slowly inject the entire volume of PMVs into the muscle and allow the animal to recover under a heat lamp.After 12 hours, sprinkle 75%ethanol over the muscle, open the skin with scissors, and harvest the entire gastrocnemius muscle at both ends. Rinse the muscle with PBS and place it under a fluorescence microscope. Using the 20 times objective lens, remove the non-fluorescing muscle tissue pieces with a sharp razor blade and mince the remaining strongly red fluorescing pieces into about nine cubic millimeter cubes.Embed each cube in an optimal cutting temperature medium. Then submerge the cubes in liquid nitrogen for five minutes. And store the tissue samples at minus 20 degrees Celsius until their analysis.PMVs generated from BMSCs as demonstrated predominantly exhibit a size in the range of about three micrometers in diameter as examined under phase contrast microscopy using the 20 times objective. BMSC transfection with a fluorescent expression cassette for 48 hours illustrates the cytoplasmic encapsulation of the EGFP protein by the PMVs. Further, standing with a fluorescent mitochondrial dye reveals that a large fraction of the PMVs contain mitochondria.The majority of which stain positive for a mitochondrial membrane potential dye, indicating the functionality of the mitochondria. Polyethylene amine treatment prior to their en vivo administration prevents PMV aggregation during their centrifugal concentration for cellular component removal. Membrane dye labeling of the BMSCs before PMV generation facilitates the generation of fluorescence trackable PMVs.Indeed, visualization of frozen sections of the gastrocnemius muscle after intramuscular delivery of labeled PMVs indicates PMV delivery to the muscle cell cytoplasm probably via endocytosis. When attempting the PMV generation procedure it's important to remember to load the syringe with the appropriate number of cells to depress the plunger quickly when flushing the cells through the filter. After its development, this technique paved the way for researchers in the field of stem cell therapy to explore the treatment of age related diseases in mice.After watching this video, you should have a good understanding of how to prepare PMV from BMSC for the delivery of cytoplasm into the gastrocnemius muscle of mice.

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Developmental Biology

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...