Name: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2007-02-25T00:00:00.000+00:00
Duration: 22 min 6 s
Description: Learn to derive transplantable hematopoietic stem cells from mouse embryonic stem cells using retroviral HoxB4 and OP9 stromal co-culture. This protocol enables rescue of lethally irradiated mice.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

Differentiation of Embryonic Stem Cells
<ol>
<li>Collect a near confluent flask of embryonic stem cells (ESC) via treatment with ESC trypsin.</li>
<li>Resuspend cells in 5 mL of Differentiation media and transfer to a non-gelatin coated T25. Incubate at 37&deg;C for 45 minutes. Retrieve supernatant and collect cells via centrifugation. Note: Mouse embryonic fibroblasts (MEFs) from the ESC cultures attach readily to the non-gelatin coated T25 and thus are depleted from the culture during this plating step. If you are not culturing your ESC on MEFs, you can skip the pre-plate. </li>
<li>Resuspend MEF-depleted ESC in Differentiation media at 333,333 cells/50 mL. This concentration results in 100 ESC/15 mL.</li>
<li>Using multi-channel pipettor, plate ESC at 100 cells/15 mL drop on 15 cm2 petri plates. With an 8-channel pipettor, you should be able to fit about 18-22 rows of drops per plate (about 5ml per plate). Note:For this protocol, 2-5 15 cm2 dishes of drops will be more than sufficient and should yield 2-5 x 106 day six EB-derived cells. </li>
<li>Gently flip plates to invert drops. Incubate at 37&deg;C 5% CO2 for 48 hours.</li>
<li>Pool drops by gently swirling plates and transfer to 15 mL conical tube. Rinse plates with 4-6 mL of PBS and add to same 15 mL conical. Note: You may pool up to five hanging drop plates. The EBs will grow as they continue to differentiate and if they are too concentrated they tend to form large clumps. </li>
<li>Allow pooled EBs to settle by gravity (about 10 minutes). Aspirate supernatant without disturbing EBs. Gently resuspend in 10 mL Differentiation media and transfer to 10 cm non-adherent petri plate.</li>
<li>Place petri plate on plate shaker at 50 rpm and incubate at 37&deg;C 5% CO2.</li>
<li>24 hours later (day four of differentiation), swirl plates to concentrate EBs in center of petri dish. Carefully exchange 50% of media (5 mL) with 5 mL fresh Differentiation Media. Return plate to incubator for two more days.</li>
</ol>
EB dissociation and infection with MSCV-HoxB4-IRES-GFP
<ol>
<li>On day six of differentiation, transfer EBs to 15 mL conical tube. Allow to settle by gravity.</li>
<li>Aspirate media and resuspend in 10 mL PBS. Allow EBs to resettle by gravity. Aspirate PBS.</li>
<li>Add 250 mL dissociation enzyme mix and 1 mL PBS. Incubate in 37&deg;C water bath for 20 minutes, occasionally swirling tube to mix enzymes and EBs.</li>
<li>Add 8 mL enzyme-free dissociation buffer.</li>
<li>Triturate mixture using 5 mL pipette until EBs are fully dissociated (about 10 times; excessive pipetting will increase death within the EB-derived cell preparation). <ol>
<li>Mixture will become cloudy with cells as EBs dissociate.</li>
<li>Placing pipette tip against bottom of conical tube during trituration will create a shear force that will greatly facilitate dissociation.</li>
</ol></li>
<li>Collect cells via centrifugation.</li>
<li>Resuspend EB-derived cells in 5 mL of 10% IMDM and count using trypan blue exclusion Note: Between day four and day six of differentiation, EBs cavitate which results in a large amount of apoptosis and cell death.Thus, at day six, upwards of 30% of EB-derived cells may stain with trypan blue. </li>
<li>Resuspend EB-derived cells at 100,000 cells/2 mL of 10% IMDM+viral supernatant such that an MOI of 5-10 is achieved.Add protamine sulfate to a final concentration of 8 mg/mL. Note: Prepare enough EB/viral supernatant mix to plate four 6-well plates (assuming 2 mL/well). </li>
<li>Plate 2 mL EB/viral supernatant mix per well of four 6 well plates pre-plated with OP9 stroma cells (see below).</li>
<li>Centrifuge plates at 2500 rpm at room temperature for 90 minutes. Transfer plates to incubator at 37&deg;C 5% CO2.</li>
<li>4-6 hours later, harvest supernatant from plates and collect any potential cells remaining in suspension via centrifugation.</li>
<li>Resuspend pellet (may be small) in 48 mL 10% IMDM+cytokines. Dispense 2 mL/well of resuspension to original four 6-well plates in which infection was performed and supernatant was collected. Note: Always prepare 10% IMDM+cytokines fresh at time of use. </li>
<li>Incubate plates at 37&deg;C 5% CO2 for seven days. Colonies should be apparent by day four post infection and large and well-formed by day seven. A robust expansion should yield 40-60 colonies/well on day seven.</li>
<li>On day seven post-infection, collect and pool all cells (including OP9 stroma) from all wells by treatment with trypsin. Note: DO NOT discard supernatant or PBS washes employed during trypsin treatment.At this point in the culture, some colonies may be only loosely adherent and there are many cells floating in suspension.Collect and pool all washes and supernatant to ensure that no potentially valuable cells are discarded. </li>
<li>Resuspend cells in 8 mL fresh 10% IMDM+cytokines. Distribute 2 mLs/flask into four T75 flasks. Add an additional 13 mLs 10% IMDM+cytokines to each flask. Incubate at 37&deg;C 5% CO2 for three days (a total of ten days post infection).</li>
</ol>
&nbsp;
Culture and plating of OP9 stroma
<ol>
<li>Maintain OP9 stroma according to standard protocols in 20% a-MEM. OP9 cells will change properties when grown to confluency. Always split at 80% confluency at no more than 1:3 split.</li>
<li>24 hours prior to infection of day six EB-derived cells with MSCV-HoxB4-IRES-GFP, plate 25,000 OP9 cells/well of four 6-well plates in 20% a-MEM.</li>
</ol>
Collection, fractionation and transplantation
<ol>
<li>Collect cells expanded on OP9 stroma for ten days via treatment with trypsin. Count cells. Note: DO NOT discard supernatant or PBS washes employed during trypsin treatment. At this point in the culture, some colonies may be only loosely adherent and there are many cells floating in suspension.Collect and pool all washes and supernatant to ensure that no potentially valuable cells are discarded. Note: A good expansion should yield between 40-50 x 106 cells/original 6-well plate infected, for a total of 160-200 x 106 cells. </li>
<li>Cells may be fractionated via magnetic bead selection or FACS according to standard techniques at this point in the protocol.</li>
<li>For transplantation, deliver cells via retro-orbital or tail vein injection to Rag-2/gc deficient recipients (weighing between 15-22 grams) subjected to a split dose of 9.25 gy of irradiation 2.5 hrs. apart. Note: It is CRITICAL that the weight of the animals fall between 15-22 grams at time of irradiation. If they are larger than 22 grams, 9.25 gy will not be a sufficient dose of irradiation to ensure the appropriate ablation and cells may not mediate rescue from lethal irradiation and/or engraft the hematopoietic compartment. If transplanting larger animals, the appropriate irradiation dose must be determined experimentally. </li>
<li>A minimum dose of 2-5 x 106 cells/animal is required to guarantee rescue from lethal irradiation. If fractionating cells, inject 2-5 x 106 cell equivalents (e.g. if population of interest represents 10% of unfractionated pool of cells, inject 2-5 x 105 cells/animal). Note: If injecting &gt;2 x 106 cells, ALWAYS inject via tail vein to avoid teratoma formation in orbit, which may result when high numbers of cells are delivered retro-orbitally. </li>
<li>Maintain Rag-2-/-/gc-/- deficient recipients in autoclaved cages. Depending on the &ldquo;cleanliness&rdquo; of the animal facility, post-transplant animals may require administration of acidified water or Trimethiprim-Sulfasoxazole (Septra) for the 5 weeks following lethal radiation. </li>
<li>Expect &gt;90% GFP+ ES-derived leukocytes in peripheral blood at 3 weeks post-transplant. Engraftment should be multi-lineage, although lymphocytes are known to silence retroviral HoxB4-IRES-GFP. </li>
</ol>

<table><tbody><tr><td>C57Bl/6 Rag-2/gamma-c deficient mice</td><td>Animal</td><td>Taconic Farm</td><td></td><td></td></tr><tr><td>Plate shaker</td><td>Tool</td><td>VWR international</td><td>33998-360</td><td>Orbital Shaker by Ikaworks</td></tr><tr><td>Multi-channel pipettor</td><td>Tool</td><td>VWR international</td><td>15000-174</td><td>ePet by BioHit</td></tr><tr><td>ES trypsin</td><td>Reagent</td><td>Invitrogen</td><td>15090-046</td><td>0.25% Trypsin in PBS</td></tr><tr><td>Standard trypsin</td><td>Reagent</td><td>Invitrogen</td><td>25300-062</td><td>0.05% Trypsin-EDTA</td></tr><tr><td>PBS</td><td>Buffer</td><td>Invitrogen</td><td>20012-027</td><td></td></tr><tr><td>Enzyme-free dissociation buffer</td><td>Buffer</td><td>Invitrogen</td><td>13151-014</td><td></td></tr><tr><td>Dissociation enzyme mix</td><td>Buffer</td><td>Invitrogen</td><td>17104-019</td><td>500 mg Collagenase IV</td></tr><tr><td>Hyaluronidase</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H2126</td><td>freeze in 500 &amp;mu;L aliquots and avoid repeated freezing and thawing.</td></tr><tr><td>DNAse</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D4527</td><td></td></tr><tr><td>DMEM</td><td></td><td>Invitrogen</td><td>10-017CV</td><td></td></tr><tr><td>Protamine sulfate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P3369</td><td>8 &amp;mu;g/mL</td></tr><tr><td>EB differentiation media</td><td>medium</td><td></td><td></td><td>Please view recipe on attached Protocol.doc</td></tr><tr><td>10% IMDM</td><td>medium</td><td></td><td></td><td>Please view recipe on attached Protocol.doc</td></tr><tr><td>10% IMDM+cytokines</td><td>medium</td><td></td><td></td><td>Please view recipe on attached Protocol.doc</td></tr><tr><td>20% anti-MEM</td><td></td><td></td><td></td><td>Please view recipe on attached Protocol.doc</td></tr></tbody></table>

derivation of hematopoietic stem cells from murine embryonic stem cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

First thing that we do is we take a cofluent T 25 of umsl. So I'm just gonna wash with PBS and I'm just gonna add a few drops of TR on And let that sit for a couple minutes And I can see that the cells are cutting off the plate. So it's pretty much ready.Okay, so now I'm gonna collect the trypsin eye cells and it's a good idea to trip to trigger rate your trypsin eye cells for just a moment to break up the colonies test and put them onto the UN plate. And this will just sit in the incubator for about 30 to 45 minutes. Now I'm just gonna spin down these cells to collect them and I am just gonna aspirate.So I'm just gonna resus in a few minutes so I can count this off. So I resus suspended and now I've counted and now I'm taking out an aliquot and I'm gonna delete this aliquot to 50 mils and that's gonna gimme the appropriate concentration to get a hundred cells per drop just to make sure the cells are okay. So now we have a reservoir.Just give these a little swish again, we pour these into our reservoir. Okay, so this is already programmed. There we go.Now it's pretty straight. Turn it over and just set it aside. Every couple of plates, I just sort of just tilt it a little bit just

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.

Derivation of Human Embryonic Stem Cells by Immunosurgery

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Learn to derive transplantable hematopoietic stem cells from mouse embryonic stem cells using retroviral HoxB4 and OP9 stromal co-culture. This protocol enables rescue of lethally irradiated mice.

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Explore More Videos