JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

1, 2, 1

1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles

Article
    Downloads Comments Metrics
     

    Summary

    mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

    Date Published: 6/07/2011, Issue 52; doi: 10.3791/2559

    Cite this Article

    Lynch, M. R., Gasson, J. C., Paz, H. Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny. J. Vis. Exp. (52), e2559, doi:10.3791/2559 (2011).

    Abstract

    The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

    Protocol

    1. Preparation of ES cells

    1. Culture ES cells according to standard protocol for your particular cell line. Make sure to prepare enough ES cells to plate onto of the mStrawberry OP9 cells for Day 0 (ES cells will be plated at 1x10^3 cells per cm2 at Day 0)

    2. Preparation of OP9 cells

    1. Before growing mStrawberry OP9 cells, prepare the OP9 growth media
      OP-9 Cell Culture Medium
        Stock Final conc. Volume (mL)
      aMEM     39.5
      FBS (OP-9 tested) 100% 20% 10
      L-glutamine 100X (200mM) 2mM 0.5
            50mL

      Store at 4°C and use within one week of preparation
    2. In order to maintain mStrawberry OP9 cells, media must be changed every 2 days and cells should be subcultured prior to cells reaching confluency (subculture at 70-90% confluency). mStrawberry OP9 cell cultures are never grown beyond 90 % confluency as this will result in their differentiation into adipocytes. See Figure 3.
      1. Subculture cells by washing cells 2X with PBS (or 1x with PBS + 1x 0.1% with Trypsin-EDTA)
      2. Add pre-warmed 0.1% Trypsin-EDTA at 37°C for 5 minutes (or until cells detach)
      3. When the cells have detached, add OP-9 growth medium. Transfer cells to a 50mL conical tube.
      4. Pellet cells at 300 x g for 5 minutes at room temperature. Discard supernatant.
      5. Resuspend cells in fresh mStrawberry OP9 growth media
      6. Plate cells at a minimum density of 104cells/cm2
      7. Grow mStrawberry OP9 cells to confluency starting 3 days prior to day needed for co-culturing ES cells (Day 0, Day 5, or Day8/9 of the experiment)

    3. ES/mStrawberry OP9 Co-culture

    1. Prepare Co-culture differentiation media
        Stock Final Volume (mL)
      aMEM     39.5
      FBS 100% 20% 10
      L-glutamine 200mM 2mM 0.5
            50mL
    2. mStrawberry OP9 cells (Day –3)
      1. ALWAYS Subculture OP9 cells on Day -3, so that you have confluent layers of mStrawberry OP9 cells for Day 0 of co-culture. See Figure 4b. Plate mStrawberry OP9 cells at 1x104/cm2 for confluency at Day 0. Be sure to subculture extra cells for Day 5 and Day 8/9 for the continuation of co-culture.
    3. ES/mStrawberry OP9 co-culture Set-Up (Day 0)
      1. Wash ES cells 2x with PBS
      2. Trypsinize ES cells with pre-warmed 0.25% Trypsin-EDTA, incubate for 5min @ 37°C
      3. Add Co-culture differentiation media to cells
      4. Pellet ES cells at 400xg, 5-7min, RT Discard supernatant
      5. Resuspend cells in Co-culture differentiation media
      6. Remove media from mStrawberry OP9 cells
      7. Add Co-culture differentiation media to mStrawberry OP9 flasks
      8. Plate 1x103 ES cells/cm2 to flask of mStrawberry OP9 cells. Incubate cells at 37° C, 5% CO2 with humidity.
    4. mStrawberry OP9 cells (Day 2)
      1. Subculture mStrawberry OP9 so that you have confluent layers of mStrawberry OP9 cells for Day 5 of co-culture. Plate mStrawberry OP9 cells at 1x104/cm2 for confluency at Day 5 (as in step 3.2). Be sure to subculture extra cells for Day 8/9, if necessary (as in step 2.2).
    5. ES/mStrawberry OP9 co-culture (Day 3)
      1. Carefully remove medium from co-culture and replace with fresh Co-culture differentiation medium


    6. ES/mStrawberry OP9 co-culture (Day 5)
      1. Carefully wash co-cultures 2x with PBS
      2. Trypsinize cells with pre-warmed 0.25% Trypsin-EDTA
      3. Wash cells off flask with Co-culture differentiation media
      4. Pellet cells at 400xg, 7min, RT. Discard supernatant
      5. Add Co-culture differentiation media to cells. Use a 21-gauge bluntended needle to resuspend cells and to ensure for single cell suspension
      6. Count cells using a hemocytometer. Should have 100-150 fold increase in ES cell number from Day 0.
      7. Re-seed 6.48x104/cm2 – 7.76x104/cm2 co-culture cells on to a new layer of confluent, mStrawberry OP9 cells
      8. Day 5 ASSAYS -Remaining co-culture cells can be used for flow cytometry analysis, cytopreps(30K cells), ChIP assays, etc.
    7. ES/mStrawberry OP9 co-culture (Day 8/9 and Day 12)
      1. Remove differentiation media from flask and save. Wash monolayer 1x with PBS to remove hematopoietic cells in suspension. Do not toss out hematopoietic cells.
      2. Trypsinize adherent cells with pre-warmed 0.25% Trypsin-EDTA
      3. Wash cells off flask with ES differentiation media and add to hematopoietic cells found in suspension=
      4. Pellet cells at 400xg, 7min, RT. Discard supernatant
      5. Add Co-culture differentiation media or PBS to cells. Use a 21-gauge bluntended needle to resuspend cells and to ensure for single cell suspension Count cells using a hemocytometer.
      6. Co-culture can be continued for 14 days to evaluate more mature hematopoietic progenitors by re-seeding co-culture cells onto a new layer of confluent, mStrawberry OP9 cells at Day 8 or 9 and then again on Day 12. Re- seed optimized cell number based on required cell recovery for day of assay.
    8. Evaluation of ES-derived progeny (Day 1-14). ES-derived progeny can be evaluated on any day of co-culture by flow sorting out mStrawberry negative cells (ES-derived progeny).
      1. Remove differentiation media from flask and save. Wash monolayer 1x with PBS to remove hematopoietic cells in suspension. Do not toss out hematopoietic cells.
      2. Trypsinize adherent cells with pre-warmed 0.25% Trypsin-EDTA
      3. Wash cells off flask with Co-culture differentiation media and add to hematopoietic cells found in suspension
      4. Pellet cells at 400xg, 7min, RT. Discard supernatant
      5. Resuspend cells with 21-gauge bluntended needle and count
      6. ES-derived progeny (mStrawberry negative cells) can be flow sorted from mStrawberry positive OP9 cells. ES-derived progeny can then be characterized by flow cytometry analysis, cytopreps, ChIP assays, etc.

    4. Representative Results:

    Figure 1
    Figure 1. ES/mStrawberry OP9 Co-culture System. Our lab uses an in vitro ES cell co-culture system to model hematopoietic development. In this co-culture system, ES cells differentiate, into hemangioblasts at Day5, when placed on a confluent layer of mStrawberry OP9 cells. As the co-culture proceeds, hematopoietic precursors are present at Day 8, while terminally differentiated hematopoietic lineages appear by Day 14. Arrows indicate days of differentiation when passaging of ES-derived progeny onto new confluent layers of mStrawberry OP9 cells in necessary.

    Figure 2
    Figure 2. Proper ES cell Differentiation. ES cells are seeded onto a confluent layer of mStrawberry OP9 at Day 0. ES-derived progeny begin to become visible at Day 3 of differentiation, with ES-derived progeny looking like a cluster of cells with a defined border or beginning to form a whorl pattern. Hemangioblasts, which are the common mesodermal precursor for the endothelial and hematopoietic lineages, should appear as piled up whorls of cells at Day 5. For co-culture periods longer than five days, ES-derived progeny should appear as clusters of hematopoietic cells. Two to four celled clusters, at Day 6/7, should appear and grow into larger cell clusters by Day 8/9 of co-culture. Other ES derived progeny are also found tightly bound to the adherent stromal cell layer.

    Figure 3
    Figure 3. Unsuccessful ES cell differentiation. Results from this co-culture reflect improper ES cell differentiation. Note how the ES-derived progeny do not appear in a whorl pattern at Day 5. The absence of cells in a whorl pattern indicates improper hemangioblast formation. If hemangioblast do not develop properly, then continued co-culture will result in stunted hematopoietic lineage differentiation. Co-cultures with improper hemangioblast formation should be discarded.

    Figure 4
    Figure 4. mStrawberry OP9 cell cultures. (A) mStrawberry OP9 cell are subcultured at 70-90% confluency. (B) For proper differentiation, ES cells and ES-derived progeny are placed onto a overly confluent layer of mStrawberry OP9 cells.

    Figure 5
    Figure 5. Representative ES/mStrawberry OP9 Co-culture flow profile. All ES-derived progeny are mStrawberry negative cells and can be flow sorted from the mStrawberry OP9 cells. mStrawberry negative cell gate was determined using a traditional ES/OP9 co-culture.

    Discussion

    Proper differentiation of ES cells on confluent layer of mStrawberry OP9 cells results in the production of Flk1+ hemangioblasts at Day 5 of co-culture. Hemangioblasts should appear as piled up whorls of cells, as observed in Figure 1. For the remainder of the co-culture, visible ES-derived progeny should appear as clusters of hematopoietic cells (Figure 1). At Day6/7 two to four cell clusters should appear and grow into larger cell clusters (both adherent and in suspension) by Day 8/9 of co-culture. Other ES derived progeny are also found tightly bound within the adherent stromal cell layer.

    Although not listed in the protocol, it is critical to pre-test the FBS used in the ES growth medium, the mStrawberry OP9 growth media and the ES/mStrawberry OP9 differentiation media. Properly tested serum should ensure proper growth of mStrawberry OP9 cells, as well as, proper differentiation of ES cells in co-culture until Day 5 of differentiation. As an alternative or in addition to mStrawberry OP9 cells, OP9 cells can be irradiated, at 8000 rads, prior to seeding at confluency (7.8x104 cells/cm2). Irradiation of OP9 cells allows for the elimination of repeated replating steps, as well as, allows for almost complete elimination of old, contaminating OP9 cells from downstream assays.

    OP9 cells were engineered to express mStrawberry protein by transducing OP9 cells with 3ug/mL of a lentiviral vector expressing mStrawberry under the control of a CMV promoter (pRRLsin-CMV vector, UCLA vector core).

    Standard ES/OP9 co-culture protocols entail the placement of ES cells onto a confluent layer OP9 cells, as well as, a day 5 replating step in order to reduce old, contaminating OP9 cells from cell passage. Additionally, only hematopoietic cells found in suspension are removed for evaluation of ES-derived progeny. However, with both a replating step and the harvesting of only suspension cells one potentially misses a substantial number of the developing ES-derived hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. In order to circumvent this issue our lab used a modified co-culture, through the use of mStrawberry-expressing OP9 cells. This assures that all ES progeny are transferred to the continued co-culture at day 5, and for the harvest of all ES-derived progeny for downstream assays. This modification provides a useful tools in the evaluation and characterization of hematopoietic progeny derived from both human and mouse ES lines.

    This ES-mStrawberry OP9 co-culture model recapitulates the various stages primitive and definitive hematopoiesis. In studying the earliest stages of hematopoietic development, many people use the BL-CFC(Blast-like colony forming cell) assay, which assess the development of the hemangioblast from the ES cell. While the BL-CFC assay is a useful tool when investigating early hematopoietic development, the mStrawberry-ES co-culture systems exhibits increase utility as it allows for the assessment of hemangioblast, as well as, more adult hematopoietic lineages.

    Previous work using traditional OP9/ES and EB differentiation protocols has shown that additional factors maybe necessary, such as overexpression of HoxB4, in order to derive long-term repopulating hematopoietic stem cells in vitro. Additional work is necessary to assess whether mStrawberry negative, sorted ES-derived populations contain true hematopoietic stem cells.

    Disclosures

    No conflicts of interest declared.

    Acknowledgements

    We thank U. Ganapati for the establishment and testing of the mStrawberry OP9 cell line. Additionally, we thank H. Shafifor her assistance with the co-culture. H.P. is supported by a fellowship from the National Heart, Lung, and Blood Institute (F31HL087714) (H.P.). The content of this work is solely the responsibility of the author and does not necessarily represent the official views of the National Heart, Lung, And Blood Institute or the National Institutes of Health (NIH). The UCLA Flow Cytometry Core Facility is supported by the NIH (CA-16042 and AI-28697), the Jonsson Cancer Center, the UCLA AIDS Institute, and the UCLA School of Medicine.

    Materials

    Name Company Catalog Number Comments
    OP-9 Cell Maintenance Medium Stock Components
    aMEM Invitrogen 12571-063
    FBS Omega Scientific FB01 Pre-Tested
    L-glutamine Cellgro 25-005-C I 200nM
    Trypsin-EDTA Stem Cell Technologies 07901
    ES/OP-9 Cell Differentiation Medium Stock Components
    aMEM Invitrogen 12571-063
    FBS Hyclone SH30070 Pre-Tested
    L-glutamine Cellgro 25-005-CI 200mM
    PBS (without Ca2+ and Mg2+) Cellgro 21-031-CV
    Trypsin-EDTA Stem Cell Technologies 07901

    References

    1. Baron, M. H. Embryonic origins of mammalian hematopoiesis. Exp Hematol 31 (12), 1160 (2003).
    2. Baron, M. H. Early patterning of the mouse embryo: implications for hematopoietic commitment and differentiation. Exp Hematol 33 (9), 1015 (2005).
    3. Baron, M. H., and Fraser, S. T. The specification of early hematopoiesis in the mammal. Curr Opin Hematol 12 (3), 217 (2005).
    4. Desbaillets, I., Ziegler, U., Groscurth, P., and Gassmann, M. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol 85 (6), 645 (2000).
    5. Ganapati, U., et al. Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression. Stem Cells 25 (8), 1872 (2007).
    6. Hogan, B. Manipulating the mouse embryo : a laboratory manual. 2nd ed. (Cold Spring Harbor Laboratory Press, Plainview, N.Y.) (1994).
    7. Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M. V. Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol Cell Biol 13 (1), 473 (1993).
    8. Keller, G. M. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 7 (6), 862 (1995).
    9. Nakano, T. Lymphohematopoietic development from embryonic stem cells in vitro. Semin Immunol 7 (3), 197 (1995).
    10. Nakano, T. In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis. Int J Hematol 65 (1), 1 (1996).
    11. Nakano, T., Kodama, H., and Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265 (5175), 1098 (1994).
    12. Turksen, K. Embryonic stem cells : methods and protocols. (Humana Press, Totowa, N.J.) (2002).

    Comments

    15 Comments

    hi

    thanks for this video.
    my question..what is the different between the op9 media and differentiation media?
    Reply

    Posted by: AnonymousJune 10, 2011, 9:01 PM

    Potentially the serum. Serum needs to be tested for proper OP9, ES cell, can co-culture growth. Serums used for OP9 cell maintenance is not always appropriate for ES cell differentiation.
    Reply

    Posted by: Helicia P.June 13, 2011, 1:42 PM

    Thank you for this video.
    Would you mind please tell me how to prevent OP9 cells from differentiating into adipocyte in the co-cultures?
    Reply

    Posted by: saloomeh m.July 20, 2012, 12:10 PM

    The OP9s should differentiate in the co-cultures. This ensures good ES cell differentiation.
    Reply

    Posted by: Helicia P.July 20, 2012, 12:36 PM

    Beautiful studies! I would like to ask if we can obtain mStrawberry/Op9 cells from you. If you have, we also like to obtain OP9-DL1 cell line.
    Reply

    Posted by: Byoung K.May 14, 2013, 8:41 PM

    We appreciate your interest in the mStrawberry OP9 cells. We suspect that these cells may be passage sensitive and we are keeping them at low passage numbers. As a result, we do not have enough vials to share them with you at this time. To develop these cells, the OP9 cells were transfected with an
    m-Strawberry mutant fluorescent protein derived from DsRed, using a third-generation self-inactivating lentivirus. Also, we do not have OP9 DL1 cells.

    .
    Reply

    Posted by: m l.May 14, 2013, 10:19 PM

    I would like to kindly ask you for a piece of advice. I've been trying to coculture embryonic stem cells with the OP9 feeders, but I noticed that the ESCs don't adhere, and even at day 5 I couldn't notice any colonies. The OP9 cells that I used were 80% confluent, and I followed exactly the protocol that you described. Did you happend to experience this problem, or do you know any reasons why this could oocur?
    Thank you very much in advance.
    Reply

    Posted by: Anca R.August 13, 2013, 8:05 AM

    What type of ES cells are you using, mouse or human? Also, if you look back at the protocol, the OP9 cell layer needs to be 100% confluent before plating the ES cells on top of them. Almost to the point of being overgrown I would say works the best. Try that and see if that helps.
    Reply

    Posted by: Helicia P.August 13, 2013, 12:57 PM

    Hello, I'm trying to differentiate induced pluripotent stem cells in T cells and the middle point consists in the generation of hematopoietic progenitors, using your protocol, followed by the transfer on OP9-DL1 feeder cells in the presence of IL2. However, the yield of differentiated colonies is very low, and the iPSCs maintain their morphology. Do you have any recommendations on how to improve the protocol? Thanks a lot.
    Reply

    Posted by: Cristina R.August 28, 2013, 3:59 PM

    Would it also be possible to have the recipe for the differentiation medium, please?
    Reply

    Posted by: Cristina R.August 28, 2013, 4:55 PM

    The recipe for differentiatiom medium is in the protocol. The FBS should be tested out to at least Day5 in a co-culture Choose a batch of serum that gives the correct differentiation and cell growth.
    Reply

    Posted by: Helicia P.August 28, 2013, 6:42 PM

    We have never used iPSCs or grown the co-cultures with IL-2, so this problem may be something particular to your ES cells or something else. How many days did you let your co-cultures grow ?
    Reply

    Posted by: m l.August 28, 2013, 6:10 PM

    We have never tried to differentiate iPS cells using this protocol so not sure how well the cells differentiate. Are you saying that you the iPS cells do not differentiate into hemangioblasts?
    Reply

    Posted by: Helicia P.August 28, 2013, 6:44 PM

    There are studies showing that iPSCs also differentiate into hemangioblasts. I started working on in vitro differentiation of iPSCs 4 months ago, and so far I didn't have positive results with my cells. However, I've been using the FBS that we normally have for cell culture in the lab, maybe I should try different companies. Thanks a lot for your quick reply.
    Reply

    Posted by: Cristina R.August 29, 2013, 6:15 AM

    The recipe for differentiatiom medium is in the protocol. The FBS should be tested out to at least Day5 in a co-culture Choose a batch of serum that gives the correct differentiation and cell growth.
    Reply

    Posted by: m l.August 28, 2013, 6:06 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter