Name: identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry
Uploaded: 2017-07-17T12:30:00
Description: Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

The authors thank Prof Frederic Geissmann and Prof Christian Schulz for insightful discussions; Dr Hannah Garner for critical reading of the manuscript, Dr Xavier Montagutelli and Dr Jean Jaubert and the staff of the Institut Pasteur animal facility for support with mouse husbandry; and Pascal Dardenne and Vytaute Boreikaite, an Amgen Scholar, for their technical assistance. Research in the E.G.P. laboratory is funded by the Institut Pasteur, the CNRS, the Cercle FSER (FRM and a starting package from the Institut Pasteur and the REVIVE consortium. L.I. is supported by a PhD fellowship from the REVIVE consortium.

Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Pasteur (CETEA).
1. In Utero Pulse Labeling in Csf1r MeriCreMer Rosa LSL-YFP Embryos
<ol>
	<li>Prepare the stock solution of 4-hydroxytamoxifen (OH-TAM, 50 mg/mL).
	<ol>
		<li>Under the fumehood, open the 25 mg vial of OH-TAM and add 250 &#181;L ethanol (100%).</li>
		<li>Transfer the ...

<ol>
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The different waves of hematopoietic precursor cells partially overlap within a short time frame, which makes the analysis of the contribution of each wave of developmental hematopoiesis to immune cells technically very challenging.
Tamoxifen-inducible Cre systems offer the opportunity to tag specific cells in a temporally inducible manner and to perform lineage analysis in embryos or adults, without the need for ex vivo or in vitro culture or transplantation. In tamoxifen-in...

There are several successive but overlapping waves of hematopoietic progenitors during development whose myeloid progeny remain into adulthood. First, unipotent &#34;primitive&#34; progenitors emerge in the mouse yolk sac 1,2 between E7.5-E8.25 and give rise to embryonic macrophages without any monocytic intermediate. Whether macrophages derived from primitive progenitors persist in the adult brain as microglia remains a subject of active investigation. Second, Erythro-Myeloid Precursors (EMPs) arise in the yolk sac at E8.5, enter the bloodstream and colonize the embryo. EMPs emerge from the yolk sac hemogenic endothelium in a Runx1-dependant endothelial-to-hematopoietic transition 3,4. While EMP can differentiate into macrophages within the yolk sac, they also colonize the fetal liver from embryonic day (E)95 and differentiate into erythrocytes, megakaryocytes, macrophages, monocytes granulocytes and mast cells 6. The macrophages that derive from EMPs exhibit proliferative capacity in developmental and adult tissues. Whether EMP-derived macrophages bypass the monocyte stage of differentiation is still controversial as very little is known about their differentiation pathway 7,8. Finally, Hematopoietic Stem Cells (HSCs) emerge at E10.5 within the embryo proper from the aorta-gonad-mesonephros region and migrate to the fetal liver. HSCs with long-term repopulation capacity are only detected after E11 (at the 42 somite pair stage)9. There, they expand and differentiate from E12.5 until definitive hematopoiesis begins to shift to the bone marrow, which becomes the predominant site of blood cell production for the duration of postnatal life 10.
The spatial and temporal overlap in emergence, as well as shared immuno-phenotypic markers has thus far hampered our ability to distinguish the specific contributions of these waves of embryonic hematopoietic progenitors. While both EMP and HSC are generated in a Runx1-dependent manner and express the transcription factor Myb and the growth factor receptor Csf1r (Colony-Stimulating Factor 1 Receptor, also known as Macrophage Colony Stimulating Factor Receptor) among others, EMPs can be distinguished from HSCs by their lack of lymphoid potential, both in vitro and in vivo, their lack of long-term repopulating potential and lack of surface expression of the lineage marker Sca-1 11. Genetic fate mapping models are required to characterize macrophage ontogeny since they allow targeting embryonic progenitors in a cell-specific and time-specific manner. Here we present the fate-mapping protocol used in our laboratory to discriminate between the two lineages of macrophages found in most adult tissues: HSC-derived infiltrating macrophages and HSC-independent resident macrophages.
Tissue resident macrophages have been traced back to Myb-independent precursor cells expressing the cytokine receptor Csf1r12 and are present in the embryo at E8.5-E10.5 using three complementary fate-mapping strategies 6. In order to study yolk sac hematopoiesis without labeling fetal HSCs, we use a transgenic strain, Csf1rMeriCreMer, expressing a tamoxifen-inducible fusion protein of &#39;improved&#39; Cre recombinase and two mouse estrogen receptors (Mer-iCre-Mer) under the control of the Csf1r promoter. Hence, the Cre recombinase will be active in Csf1r-expressing cells during a limited time-window. When used with a reporter strain containing a fluorescent protein downstream of a lox-STOP-lox cassette (Rosa26LSL-eYFP), it will lead to the permanent genetic labeling of the cells present at the time of induction but also of their progeny. Administration at E8.5 of the active form of tamoxifen, 4-hydroxytamoxifen (OH-TAM), labels EMPs and macrophages, without labeling yolk sac unipotent &#34;primitive&#34; progenitors or fetal HSCs. Thereby, we have characterized the immunophenotype of EMPs and their progeny during embryonic development, as well as assessed the contribution of yolk sac-derived macrophages to the adult macrophage pools. Further work is required to characterize whether primitive progenitor-derived macrophages are also labeled using this approach and whether they can contribute to adult macrophage pools.

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			<td>#5 Straight Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
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			<td>MC19/B Pascheff-Wolff Spring Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>15371-92</td>
			<td></td>
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		<tr>
			<td>8.5 cm straight scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
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		<tr>
			<td>Anti-Mouse Kit-PE (clone 2B8)</td>
			<td>BD Pharmingen</td>
			<td>553355</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td>
			<td>Sony</td>
			<td>1149120</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td>
			<td>eBioscience</td>
			<td>17-5892</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td>
			<td>Biolegend</td>
			<td>560512</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80-BV421 (clone BM8)</td>
			<td>BD Pharmingen</td>
			<td>123137</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td>
			<td>BD Pharmingen</td>
			<td>552850</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
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			<td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
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			<td>PBS 1x</td>
			<td>Fischer Scientific</td>
			<td>12559069</td>
			<td></td>
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			<td>Fetal Bovine Serum</td>
			<td>Fischer Scientific</td>
			<td>11570506</td>
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			<td>Collagenase D&#160;</td>
			<td>Roche</td>
			<td>11088882001</td>
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			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527-20KU</td>
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			<td>6-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353046</td>
			<td></td>
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		<tr>
			<td>12-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353224</td>
			<td></td>
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			<td>24-well tissue culture plate</td>
			<td>Fischer Scientific</td>
			<td>11874235</td>
			<td></td>
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		<tr>
			<td>96 wells U-shape bottom tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353227</td>
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			<td>&#160;Syringe Plastipak 2 mL&#160;</td>
			<td>Dutscher Dominique</td>
			<td>300185</td>
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			<td>Nylon grid 100 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352360</td>
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			<td>Nylon grid 70 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352350</td>
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			<td>15 ml Falcon tubes</td>
			<td>Dutscher Dominique</td>
			<td>352096</td>
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			<td>Stericup GP Millipore filtration kit, 0.2 &#956;m</td>
			<td>Dutscher Dominique</td>
			<td>51246</td>
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			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906-500G</td>
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			<td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td>
			<td>Sigma</td>
			<td>H7904-25MG</td>
			<td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td>
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			<td>EDTA</td>
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			<td>E9884-500G</td>
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			<td>DAPI, 1 ML (1 MG/ML IN WATER)</td>
			<td>Fischer Scientific</td>
			<td>10116287</td>
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			<td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>B75408</td>
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			<td>CytoFLEX Daily QC Fluorospheres</td>
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			<td>VersaComp Antibody Capture Bead kit (2x5 mL)</td>
			<td>Beckman Coulter&#160;</td>
			<td>B22804</td>
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			<td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td>
			<td>The Jackson laboratory</td>
			<td>19098</td>
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			<td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td>
			<td>The Jackson laboratory</td>
			<td>6148</td>
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identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

Genetic fate mapping was achieved by administration at E8.5 of OH-TAM into Csf1r-Mer-iCre-Mer females mated with males carrying a Rosa26-LSL-eYFP reporter. In the presence of OH-TAM, excision of the stop cassette leads to the permanent expression of YFP in the cells expressing Csf1r. We collected two hematopoietic tissues: the yolk sac and the fetal liver and a non-hematopoietic tissue, the neuroectoderm, from the embryos at E10.5. Single-cell suspensions were obtained by enzymat...

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

The overall goal of this experiment is to characterize yolk sac hematopoietic progenitors and their progeny in vivo using a mouse tamoxifen induced fate mapping system and flow cytometry. This can help answer key questions in the immunology field concerning the development and differentiation of hematopoietic progenitors that are distinct from hematopoietic stem cells. The main advantage of this technique is that the hematopoietic progenitors labeled within a limited time window and the progeny such as resident macrophages can be monitored during the development and adulthood.Generally, individuals new to this method will struggle because of the deal of cells for embryonic tissue and the large volume of the simultaneously treated samples. To begin the experiment, first transfer dissected mouse uterine horns into a 10 millimeter petri dish filled with approximately 30 milliliters of ice cold PBS. Immediately after, grasp the uterus muscle layers at the cervical end of the horn and slide fine scissors between the muscle layer and a decidual tissue to release the embryos.Then with fine forceps remove Reichert's membrane and remove the placenta. Gently dissect the yolk sac and transfer it to a 24-weld tissue culture plate filled with 0.5 milliliters of the digestion solution. After severing the umbilical and vituline vessels immediately transfer the embryo into a 12-weld tissue culture plate filled with 2 milliliters of 10 millimolar ice cold EDTA.After removing the head from the embryo with sharp fine scissors, incubate the body and the head on ice for 10 to 15 minutes and then remove the embryo and collect the EDTA containing the blood. Next, remove the amnion surrounding the embryo. Afterward remove the hind limbs and foreign limbs from the embryo.Using a pair of fine forceps, open the thorax. Then, grasp the anterior of the heart and gently pull the tissue while releasing the internal organs from the body with a second pair of forceps. Under a dissecting microscope, separate the fetal liver from the heart and gut.And transfer the organ to a 24 well plate filled with 0.5 milliliters of the digestion solution. Anatomical connection with the cardiovascular system is critical for fetal liver identification. The fetal liver is still in this developmental stage and the younger the embryos are, the more difficult the dissection can be.To prepare primary cell suspensions, incubate the collected organs in the digestion solution at 37 degrees Celsius for 30 minutes. Load the tissue in the enzymatic solution into a 10 micrometer strainer installed over one weld of a six-weld tissue culture plate filled with 1 milliliter of FACS buffer. Use a black rubber piston from a 2 millimeter sysringe to dissociate the tissue by gently mashing to obtain a single cell suspension.Then, aspirate the cell suspension with a Pasteur pipette and transfer it into a 15 milliliter tube. Centrifuge the suspension at 320 times G at 4 degrees Celsius for 7 minutes. After centrifugation, aspirate and discard the supernatant.Next, prepare fresh Fc blocking buffer and the resuspend the pellet in 60 microliters. Then, transfer 50 microliters of the single cell suspension into a well of a 96-well round bottom plate. Incubate the plate on ice for at least 15 minutes.Next, transfer the remaining 10 microliters of the suspension into a 5 milliliter polystyrene FACS tube to obtain a pool of samples derived from different tissues. Transfer 50 microliters of the pool for FMO controls for each fluorochrome to the 96 multi well plate. To proceed with antigen surface staining, use a set of six flourochrome antibodies to prepare an antibody solution and FACS buffer.Then prepare six antibody solutions and FACS buffer used for the FMO control staining. Afterward add 50 microliters of the antibody solutions to the samples in the 96 multi well plate. Mix the samples by gentle double pipetting and then incubate the plate on ice for 30 minutes.Centrifuge the 96 multi well plate at 320 times G at 4 degrees Celsius for 7 minutes and remove the supernatant. Wash the cells in 200 microliters of FACS buffer and spin the plate one more time. Special care should be taken when removing the supernatant by plate inversion.The 96 multi-well plate must be inverted with a single sharp wrist motion to avoid loosening the cells. Finally, using a 70 micro strainer, filter the tissue and control samples into 6 millimeter polystyrene tubes. Flow cytometric analysis can now be carried out.A population of a live non-erythrocytic cytic single cells was analyzed for the expression of the progenitor marker kit and the hematopoietic cell marker CD45. Within the targeted population, three different cell sub populations were distinguished. KIT positive CD45 negative, KIT positive CD45 low and KIT negative CD45 positive.Among the yolk sac kit positive progenitors, both CD45 negative and CD45 low cell populations contained AA4.1 positive cells that express the yellow fluorescent protein or YFP, indicating that immature KIT positive progenitors first express AA4.1 irrespective of CD45 expression. AA4.1 positive, YFP positive cells in the liver and the brain were only found in the KIT positive CD45 low progenitor population and correspond most likely to circulating progenitors that originated from the yolk sac progenitors. Macrophages were found in the kit negative CD45 positive population and were identified as F480 bright CD11 B positive cells.Macrophages in the yolk sac, liver and brain were efficiently labeled by a single 4-hydroxy tamoxifen administration that may indicate their ENP origin. Once mastered, the procedure for cell isolation and staining can be done in three to four hours if it's performed properly. While attempting this procedure, it's important to remember to always keep samples on ice as soon as they are collected.Fresh FACS buffers should be prepared daily and filtered. Following this procedure, other mash reporter or flux strains like Rosa MTMG or Rosa confetti lines can be used in order to answer additional questions on the indifferentiation and proto phase potential of the post labeled cells.

identification-erythromyeloid-progenitors-their-progeny-mouse-embryo

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

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 ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

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 ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...