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

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Fred Hutchinson Cancer Research Center.

1. Preparation of AGM-EC Monolayers for Co-culture

1. 24 h prior to the AGM dissection and sort, make  AGM-EC culture media and filter sterilize.
2. Add 0.1% gelatin in water (100 µL/well) to each well of a 96-well plate and incubate at room temperature for 15 min. Aspirate the gelatin and leave the plate in a tissue culture hood with the lid removed until the wells are dry.
3. Dissociate the AGM-EC monolayers and plate to 96-well plates.
   NOTE: Maintain the AGM-EC, prepared as previously described¹⁶, in AGM-EC culture media. For consistency, AGM-EC cell lines should be frozen as soon as sufficient numbers are obtained (generally passage 1-3) and used at a defined passage number (generally passage 5-12) for co-culture experiments. AGM-EC cell lines are derived from C57BL/6J (CD45.2) AGM. See the Table of Materials for all media compositions.
   1. Aspirate the media from the AGM-EC culture in a 75 cm² flask and add 5 mL PBS. Aspirate PBS, and add 3 mL trypsin solution (see Table of Materials).
   2. Incubate at 37 °C for 2-4 min until most cells are lifting off the plate. Add 7 mL of AGM-EC culture media and pipet 8-10 times with a 10 mL pipette to prepare single cells suspension, and transfer to a 15 mL tube.
   3. Spin at 300 x g for 5 min, remove the supernatant, re-suspend in 10 mL AGM-EC culture media, and estimate the cell number with a hemocytometer. Dilute the cells to a concentration of 1 x 10⁵ cells/mL in AGM-EC culture media.
4. Add 100 µL AGM-EC cell suspension (1 x 10⁴ cells) to each well of a gelatinized 96-well plate. Place in a 37 °C, 5% CO₂ tissue culture incubator overnight to allow the AGM-EC to attach and form a confluent monolayer.
   NOTE: Evenly distribute the AGM-EC stromal cells so there is limited overgrowth or cell clumping for optimal co-culture.
5. Prepare the AGM-EC monolayer for AGM co-culture.
   1. The following morning, prepare the AGM serum-free media.
   2. Using a 12-well multichannel pipette, remove the AGM-EC culture media from the AGM-EC and add 200 µL/well of AGM serum-free media without cytokines to wash out any residual serum-containing media.
   3. Remove the media and add 200 µL/well of AGM serum-free media with cytokines. Work quickly when removing and adding the media so that the endothelial layer is not compromised; e.g., remove and re-add media one row at a time. Place the 96-well plates in a tissue culture incubator at 37 °C until the embryonic cells are ready for sorting.

2. Preparation of Single Cell Suspension from Murine Embryonic Tissues

1. Dissect the AGM from timed matings.
   1. Set up timed matings for generating embryonic tissues of the desired age.
   2. Harvest embryos from pregnant females at 9.5 to 11.5 days post coitum (dpc), depending on the stage of pre-HSC to be analyzed.
      NOTE: Embryonic tissues are harvested from C57BL/6J (CD45.2) mice as previously described and precisely staged based on somite count¹⁶. We have focused on analysis of populations from the embryonic AGM region and its precursor, the P-Sp, between E9.5 to E11.5, based on somite staging.
   3. Dissect the AGM from the embryos²⁰.
      NOTE: Detailed protocols for the dissection of AGM from murine embryos have been published by others²⁰. Alternatively, yolk sac or other tissues purported to have pre-HSC activity could also be analyzed by this method.
2. Dissociate the dissected embryonic tissues.
   1. Collect the dissected tissues in a 15 mL conical tube containing 10 mL PBS with 10% FBS on ice. Gravity settle tissues, aspirate the PBS/FBS, and then immediately add 1 mL of 0.25% collagenase and place in a 37 °C water bath for 25 min.
   2. Add 1 mL of PBS/10% FBS and pipet about 20-30 times with a 1 mL pipette tip to obtain a single cell suspension. Add an additional 8 mL of PBS/10% FBS and centrifuge cells at 300 x g for 5 min. Discard the supernatant.

3. Antibody Staining of Murine Embryonic Cells

1. Prepare the blocking buffer for antibody staining.
   1. Add 10 µg/mL anti-mouse CD16/CD32 (Fc receptor (FcR) block) and 1 µg/mL DAPI (1 mg/mL stock in H₂O) to 1 mL PBS with 10% FBS.
   2. Draw up in a 3 mL syringe and pass through a 0.22 µm syringe filter to sterilize. Re-suspend the cell pellet from the dissociated murine embryonic tissues (from step 2.2.2) in 500 µL blocking buffer and incubate on ice for 5 min.
2. Prepare the antibody mix¹⁷.
   1. Add 10 µg/mL FcR block to 1 mL PBS with 10% FBS and 10 µL of each fluorochrome-conjugated anti-VE-Cadherin antibody (CD144, see the Table of Materials) and fluorochrome-conjugated anti-EPCR antibody (CD201, see the Table of Materials). Use a 1:100 final dilution of antibodies unless otherwise indicated based on the manufacturer's recommendations or according to titrations.
      NOTE: This antibody combination is used for defining gates for sorting, as described below (step 4.2). Sorting based on the co-expression of VE-Cadherin and EPCR enables significant enrichment for the portion of AGM-derived cells containing HSC precursor activity, as previously described¹⁷.
   2. Add additional fluorochrome-conjugated antibodies for further phenotypic analysis of individual index-sorted precursors.
      NOTE: These antibodies are not necessarily used for defining gates for sorting. In this sample protocol, we have included PE-conjugated anti-CD41 antibody and FITC-conjugated anti-CD45 antibody to analyze the relative expression of these hematopoietic markers, which evolve during the emergence of pre-HSC from hemogenic endothelium^4,5,16,17. Other markers of interest can be included as desired. Samples stained with fluorochrome-conjugated isotype antibody controls are used to define negative and positive gates for analysis.
   3. Draw up the antibody mix in a 3 mL syringe and pass through a 0.22 µm syringe filter to sterilize. Add 500 µL of the antibody mix to the cell suspension in blocking buffer, pipette to mix, and incubate on ice for at least 15-20 min.
3. Wash the stained cells.
   1. Add 8 mL PBS/10% FBS. Centrifuge the cells at 300 x g for 5 min, aspirate, and re-suspend the cell pellet with 1 mL PBS/10% FBS.
   2. Remove cell clumps by pipetting the cell suspension through a 35 μm cell strainer cap on a 5 mL tube. Wash the cell strainer with an additional 3 mL PBS/10% FBS. Centrifuge the cells at 300 x g for 5 min, aspirate, re-suspend in 500 µL PBS/10% FBS, and place on ice until FACS.

4. Index Sorting of Single Hemogenic Precursors to 96-wells with AGM-EC Stroma for Co-culture

1. Prepare the FACS machine for plate mode and align for sorting to 96-well plates according to the manufacturer's instructions. Select single cell mode and index sorting mode in the software for maximal purity mask settings and to enable acquisition of index parameters for each sorted cell.
2. Acquire a sample of cells from the antibody stained samples to set gates for sorting.
   1. Load the stained cell sample on the FACS machine and acquire cells at the lowest flow rate. Plot FSC-A verses SSC-A and select a gate that includes cells of varying size (Figure 1B) (which is based on the observation that pre-HSC activity is identified in cells of different size profiles in the AGM¹⁷). Plot FSC-A verses FSC-W and SSC-A verses SSC-W, and select gates to exclude doublets. Then plot FSC-A verses DAPI to gate live cells (negative for DAPI) (Figure 1B).
   2. Plot PECy7 (or the relevant fluorochrome for the VE-Cadherin stain) versus PerCP (or the relevant fluorochrome for the EPCR stain). Gate cells that are positive for VE-Cadherin (which includes a mixed population of endothelial cells as well as hematopoietic progenitors and pre-HSC from the AGM) and that have high EPCR expression (which enriches for pre-HSC from P-Sp/AGM¹⁷). This is the final gate that will be used to index sort single cells in step 4.3 (Figure 1B).
   3. Plot additional fluorochromes used for staining.
      NOTE: Depending on the subpopulation to be analyzed, additional gates can be set based on the detection of other markers included for staining cells. However, index sorting enables the recording of the fluorescent parameters for each sorted cell such that these parameters can be retrospectively analyzed. Thus, no additional gating is necessary at this point. For all FACS setups, samples stained with single antibodies should be used to adjust compensation, to account for spillover in the emission of overlapping fluorochromes, and with isotype control antibodies, to account for background staining and determine negative/positive gates.
3. Index sort the AGM cells.
   1. Place a 96-well plate prepared with the AGM-EC stroma in AGM serum-free media with cytokines (from step 1.5.3) in the plate block of the FACs sorting machine. Load the sample and begin sample acquisition. Select index sorting/single cell mode and begin sortingsingle cells to individual 96-wells. Use the lowest flow rate to minimize shear stress on embryonic cells.
   2. Ensure that all events are recorded during the index sorting. This will enable enumeration of the total number of cells analyzed in the sorting gate relative to the actual number sorted to individual 96-wells, as not all events recorded will be sorted to wells.
   3. Once the cells have been sorted to an entire 96-well plate, place the plate in a tissue culture incubator at 37 °C set at 5% CO₂. Repeat for additional plates required per experiment.
   4. Co-culture individually sorted cells in 96-wells containing AGM-EC (from step 4.3) for up to 7 days (5 days for cells sorted from E11-11.5 embryos, 6 days from E10-10.5 embryos, 7 days from E9-9.5 embryos). Visualize hematopoietic colonies of various sizes and morphologies with an inverted microscope at 100X magnification (Figure 2B) after the co-culture period.
      NOTE: The media does not need to be changed during the co-culture period. Sorted hemogenic precursors may initially integrate into the AGM-EC layer with an EC-like morphology and cannot be initially distinguished from AGM-EC stroma (Figure 2A). However, following 24-48 h in co-culture, small, rounded hematopoietic cells or clusters of cells may be detected. At the end of co-culture (5-7 days), individual colonies of hematopoietic cells can be visualized in the subset of 96-wells that received a sorted cell with clonal hematopoietic potential. If visually inspected wells contain more than one distinct colony, then this sample is excluded from downstream analysis to exclude samples that may have received more than one cell sorted per well.

5. Analysis of Clonal Hematopoietic Progeny Following Co-culture

1. Harvest clones from each 96-well for FACS analysis.
   1. Vigorously pipet to dissociate hematopoietic cells from endothelial layers using a multichannel pipette with 200 µL pipette tips. Try not to dissociate the endothelial layer. Remove 100 µL (~50% of the sample) for phenotypic analysis of hematopoietic progeny by FACS.
   2. Transfer to a 96-well V-bottom plate and centrifuge at 300 x g for 2 min to pellet the cells. Remove the media by flicking media from the plate with a single rapid motion while the plate is inverted (flick plate).
   3. Place the remaining 100 µL cells in the original 96-well plate in a 37 °C incubatorfor use in the subsequent engraftment assay (step 6).
2. Incubate the cells with the appropriate antibodies for hematopoietic analysis.
   1. Re-suspend the cell pellets in each well of a 96-well V-bottom with 50 µL of PBS/2% FBS containing 10 µg/mL FcR block and 1 µg/mL DAPI. Incubate the plate at 4 °C for 5 min.
   2. Add 50 µL of PBS/2% FBS containing 10 µg/mL FcR block and an antibody mix for HSC analysis containing PE-Cyanine7-conjugated anti-VE-Cadherin, PerCP-conjugated anti-CD45, PE-conjugated anti-EPCR, APC-conjugated anti-Sca1, and FITC-conjugated anti-Gr1 and anti-F4/80 (each antibody at 1:100 final dilution unless otherwise indicated based on the manufacturer's recommendations or according to titrations). Incubate the plate at 4 °C for at least 20 min.
3. Remove unbound antibody with sequential dilutions and analyze the cells.
   1. Add 200 µL/well PBS/2% FBS and centrifuge at 300 x g for 2 min to pellet the cells. Then, flick plate to discard the supernatant and add 200 µL PBS/2% FBS to each cell pellet and centrifuge at 300 x g for 2 min to pellet cells.
   2. Flick plate to discard the supernatant and add 50 µL/well PBS/2% FBS for analysis. Proceed to analyze cells by FACS using a flow cytometer equipped with a plate reader.
      NOTE: Acquire a fixed volume of cells (e.g., 35 µL of the total 50 µL used for re-suspension) to enumerate the subsets of hematopoietic progeny.
4. Analyze the FACS data for each well containing hematopoietic progeny to screen for colonies that contain HSC potential (Figure 3).
   1. Gate first the CD45 positive population that is negative for myeloid markers Gr1 and F480. Do not gate out cells that still express low levels of VE-Cadherin. Cells from the AGM-EC layer that are disrupted during harvest of hematopoietic colonies are VE-Cadherin positive and CD45 negative.
   2. Gate CD45⁺VE-Cad^-/lowGr1^–F4/80^– cells further on the subset that express high levels of EPCR and Sca1 (Figure 3A – C).
      NOTE: In prior studies, colonies lacking cells with CD45⁺VE-Cad^-/lowGr1^–F4/80^–Sca1^hiEPCR^hi phenotype (Figure 3D) reproducibly failed to provide detectable multilineage peripheral blood engraftment in irradiated recipient mice¹⁷ (data not shown); thus, these colonies need not be included in the subsequent transplantation analysis in step 6.

6. Analysis of Engraftment Properties of Individual Clones and Correlation with Phenotypic Properties Elucidated by Index Sorting

1. The day of (if possible) or the morning following the phenotypic analysis in step 5, re-suspended the remaining 100 µL cells from each 96-well containing hematopoietic colonies following co-culture (from step 5.1) by vigorous pipetting using a 200 µL pipette tip.
2. Prepare and add rescue cells from whole bone marrow of congenic strain B6.SJL-Ptprc^aPepc^b/BoyJ (B6 CD45.1) adult mice^16,17.
   1. Count nucleated bone marrow cells in Turks solution under a hemocytometer. Dilute to a concentration of 5 x 10⁵ nucleated cells/mL in PBS with 2% FBS.
   2. Add 100 µL of rescue bone marrow cells (5 x 10⁴) to each well containing hematopoietic progeny for transplantation analysis and mix by pipetting.
3. Transplant progeny of individual clones into a single lethally irradiated recipient congenic strain B6.SJL-Ptprc^aPepc^b/BoyJ (B6 CD45.1) adult mouse.
   1. Lethally irradiate the same number of B6 CD45.1 mice as the number of clones to individually inject progeny of a single clone (using 1,000 cGy from a Cesium source).
   2. Draw 200 µL total cell suspension (this includes cells from the clone as well as 5 x 10⁴ B6 CD45.1 rescue bone marrow cells) into a ½ mL insulin syringe with a 29G ½ inch needle.
   3. Lethally irradiate B6 CD45.1 adult recipients 2-24 h prior to injection.
   4. Using a plastic mouse restrainer that allows tail access, inject via the tail vein 200 µL volume containing both cells from each clone and 5 x 10⁴ B6 CD45.1 adult bone marrow rescue cells to aid in recipient survival.
   5. Ear tag and weigh the recipient mice, and check their weight/health at regular intervals (e.g., 3-4 times/week post irradiation/transplant, or per individual institutional standard operating procedures) to ensure good health.
4. Perform analysis of peripheral blood engraftment at regular intervals (e.g., 2, 16, 24 weeks) following transplantation.
   1. Remove 50-100 µL of blood via retro-orbital bleed or another ethically approved blood letting method.
   2. Add 3 mL lysis buffer (16.6 g NH₄Cl, 2 g NaHCO₃ and 74.4 mg EDTA to 2 L H₂O) to blood and incubate at room temperature for 5 min. Centrifuge at 300 x g for 5 min. Aspirate and re-suspend the pellet with 2 mL PBS/2% FBS. Centrifuge at 300 x g for 5 min.
   3. Re-suspend the pellet with 150 µL PBS/2% FBS containing 10 µg/mL FcR block and 1 µg/mL DAPI, and dispense 1/3 of the volume to a well containing 50 µL isotype controls for donor and lineage, 1/3 to a well containing 50 µL antibody for donor and isotype controls for lineage, and 1/3 to a well containing 50 µL antibodies to detect both donor and lineage.
      NOTE: Antibodies for detection of multilineage engraftment: APCeFluor780-conjugated anti-CD45.2, Pe-Cyanine7-conjugated anti-CD45.1, FITC-conjugated anti-CD3, PE-conjugated anti-F4/80, PerCP-conjugated anti-Gr1, and APC-conjugated anti-CD19, or relevant isotype controls.
   4. Identify clones with long-term engraftment by assessing peripheral blood contribution by FACS over time of CD45.2 (donor)-derived hematopoietic cells to myeloid (Gr1 and/or F4/80 positive), B-cell (CD19 positive), and T-cell (CD3 positive) (Figure 4A-B).
      NOTE: Hematopoietic engraftment can also be analyzed by CD45.2 (donor)-derived contribution to hematopoietic populations from tissues harvested from bone marrow, spleen, thymus, and peritoneum, or following secondary transplantation of bone marrow cells into B6 CD45.1 mice to analyze serial engraftment properties¹⁷.
5. Correlate each clone's phenotypic properties as determined by initial single cell index sorting with its engraftment properties. Using flow analysis software, upload sorting parameters for each index-sorted cell (correlated to the 96-well to which it was sorted). Map functional data onto flow plots to evaluate the phenotypic properties of single cells as they relate to their functional output (for example, long-term, multilineage engraftment) (Figure 4C).