Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.
Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.
Normal differentiation of hematopoietic stem cells (HSCs) is critical for maintenance of physiological levels of all blood cell lineages. During differentiation, in a coordinated response to extracellular cues including growth factors and cytokines, HSCs first give rise to multipotent progenitor (MPP) cells that have lympho-myeloid potential1,2,3,4 (Figure 1). MPPs give rise to common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) that are lineage-restricted. CLPs differentiate into the lymphoid lineages comprised of B, T, and natural killer cells. CMPs generate the myeloid lineages through two more restricted progenitor populations, megakaryocyte erythroid progenitors (MEPs), and granulocyte monocyte progenitors (GMPs). MEPs give rise to megakaryocytes and erythrocytes, whereas GMPs give rise to granulocytes and monocytes. In addition to arising through CMPs, megakaryocytes have been reported to also arise directly from HSCs or early MPPs via non-canonical pathways5,6.
Hematopoietic stem and progenitor cells (HSPCs) are characterized by the surface marker CD34 and the lack of lineage specific markers (Lin–). Other surface markers that are commonly employed to distinguish HSCs and myeloid progenitor populations include CD38, CD45RA, and CD1232 (Figure 1). HSCs and MPPs are Lin–/CD34+/CD38– and Lin–/CD34+/CD38+, respectively. Myeloid committed progenitor populations are distinguished by the presence or absence of CD45RA and CD123. CMPs are Lin–/CD34+/CD38+/CD45RA–/CD123lo, GMPs are Lin–/CD34+/CD38+/CD45RA+/CD123lo, and MEPs are Lin–/CD34+/CD38+/CD45RA–/CD123–.
The total population of CD34+ stem and progenitor cells can be obtained from human umbilical cord blood (UCB), bone marrow, and peripheral blood. CD34+ cells constitute 0.02% to 1.46% of total mononuclear cells (MNCs) in human UCB, whereas their percentage varies between 0.5% and 5.3% in bone marrow and is much lower at ~0.01% in peripheral blood7,8,9. The proliferative capacity and differentiation potential of UCB derived CD34+ cells is significantly higher than that of bone marrow or peripheral blood cells1,10, thereby offering a distinct advantage for obtaining sufficient material for molecular analyses in combination with performing immunophenotypic and morphological characterization of the cells during differentiation.
Ex vivo differentiation of umbilical cord blood derived CD34+ HSPCs is a widely applied model for investigating normal hematopoiesis and hematopoietic disease mechanisms. When cultured with the appropriate cytokines, the UCB CD34+ HSPCs can be induced to differentiate along the myeloid or lymphoid lineages11,12,13,14,15,16. Here, we describe protocols for isolation and immunophenotypic characterization of the CD34+ HSPCs from human UCB, and for their differentiation to myeloid lineage cells. This culture system is based on cytokine-induced differentiation of HSPCs in the presence of MS-5 stromal cells to mimic the microenvironment in bone marrow. The culture conditions cause an initial expansion of the CD34+ cells, followed by their differentiation to cells that express markers for the four myeloid lineage cells, namely granulocytes (CD66b), monocytes (CD14), megakaryocytes (CD41), and erythrocytes (CD235a). Applications of the CD34+ cell differentiation protocol include studies on molecular mechanisms regulating hematopoiesis, and investigations of the impact of myeloid disease associated mutations and small molecules on self-renewal and differentiation of HSPCs.
Human umbilical cord blood for experimentation was donated by healthy individuals after informed consent to Maricopa Integrated Health Systems (MIHS), Phoenix. The deidentified units were obtained through a Material Transfer Agreement between MIHS and the University of Arizona.
1. Reagents and Buffers
NOTE: Prepare all reagents and buffers under sterile conditions in a biological safety cabinet.
2. Isolation of Mononuclear Cells from Umbilical Cord Blood
NOTE: For the protocol described below, cord blood volumes of 90 to 100 mL were used. The isolation of CD34+ cells must be performed under sterile conditions in a biological safety cabinet.
3. Isolation of CD34+ Cells from Mononuclear Cells
4. Determination of Stem and Progenitor Populations by Flow Cytometry
5. Myeloid Differentiation of the CD34+ Hematopoietic Stem and Progenitor Cells
NOTE: To differentiate the CD34+ HSPCs to the myeloid lineage cells, they are first stimulated in recombinant human fibronectin fragment coated plates and then seeded on a layer of MS-5 stromal cells in myeloid differentiation medium. Differentiation may be monitored each week for 3 weeks based on expression of cell surface markers specific to the four myeloid lineages. During stimulation and differentiation, all incubations are performed at 37 °C and 5% CO2 in a humidified chamber. All steps should be performed in a biological safety cabinet.
6. Assessment of Cellular Morphology
Application of the above protocols yields 5.6 (± 0.5) x 108 MNCs and 1 (± 0.3) x 106 CD34+ cells from a cord blood unit of ~100 mL. The percentage of total CD34+ cells ranges between 80-90% (Figure 2A,B). Immunophenotypic analysis by the scheme described by Manz et al.5 demonstrates that the CD34+ cells typically consist of ~20% HSCs and ~72% MPPs that are Lin–/CD34+/CD38– and Lin–/CD34+/CD38+, respectively (Figure 1 and Figure 2A). In the MPP population, the percentages of CMPs (Lin–/CD34+/CD38+/CD123lo/CD45RA–), GMPs (Lin–/CD34+/CD38+/CD123lo/CD45RA+), and MEPs (Lin–/CD34+/CD38+/CD123–/CD45RA–) are approximately 25%, 15%, and 56%, respectively (Figure 1 and Figure 2B).
During incubation of the isolated HSPCs in myeloid differentiation conditions, there is progressive loss of the CD34 marker. Immunophenotyping shows that the percentage of CD34+ cells reduces from ~90% at isolation (Figure 2A) to ~23% on day 21 (Figure 3B). Analysis of the CD34– population demonstrates a concomitant rise in the percentage of cells expressing mature myeloid lineage markers. There is an increase in the percentage of cells expressing markers for monocytes (CD34–/CD14+/CD66b–) from an average of 1.7% to 12%, for granulocytes (CD34–/CD14–/CD66b+) from 1.3% to 5.3%, for megakaryocytes (CD34–/CD41+/CD235a–) from 1.8% to 8%, and for erythroid cells (CD34–/CD41–/CD235a+) from 0.7% to 11% (Figure 3C). Examination of Wright-Giemsa stained cells demonstrates that the morphological characteristics of the cells on day 1 and day 21 are distinct. The undifferentiated cells have large round nuclei, and very little cytoplasm. On the other hand, cells from differentiated cultures exhibit characteristics of lineage cells including mature monocytes, granulocytes, and erythroblasts (Figure 4). Thus, the culture conditions described in this protocol promote differentiation of UCB CD34+ cells to the myeloid lineage cells.
Figure 1: Hematopoietic differentiation schematic. The pluripotent hematopoietic stem cell (HSC) differentiates into the multipotent progenitor (MPP), which gives rise to the common myeloid progenitor (CMP) and the common lymphoid progenitor (CLP) cells. CMPs generate two other myeloid progenitors, the granulocyte monocyte progenitors (GMPs) and the megakaryocyte erythrocyte progenitors (MEPs). Granulocytes and monocytes arise from GMPs, and erythrocytes and megakaryocytes arise from MEPs. CLPs give rise to natural killer, B, and T cells. The cell surface markers used to characterize the cell populations in this protocol are indicated. This figure has been modified from Bapat et al.11. Please click here to view a larger version of this figure.
Figure 2: Immunophenotypic analysis of the hematopoietic stem and progenitor cells. (A) Cells were gated on forward and side scatter to select a single cell population. Dead and lineage positive cells were eliminated by staining with 7-AAD and antibodies to CD3, CD7, CD10, CD11b, CD19, and CD235a (all FITC stained). The Lin–/live cells were analyzed with antibodies to CD34 (APC-Cy7), CD38 (PE), CD123 (APC), and CD45RA (PE-Cy7). The progenitors were distinguished from the CD34+/CD38+ cells. CMPs are CD123lo/CD45RA–, GMPs are CD123lo/CD45RA+ and MEPs are CD123–/CD45RA–. Representative scatter plots from an experiment are shown. (B) Percentages of total HSPCs (total CD34+ cells), CMPs, GMPs, and MEPs in cord blood are represented. Data presented are averages with standard error from at least three independent experiments. Please click here to view a larger version of this figure.
Figure 3: Immunophenotypic analysis of myeloid lineage cells. Single cells were gated based on forward and side scatter. CD34– cells were selected by staining with antibody to CD34 (APC-Cy7). Myeloid lineages in the CD34– population were analyzed with antibodies to CD14 (PE), CD66b (PE-Cy7), CD41 (PerCP-Cy5.5), and CD235a (APC) on day 1 (A) and day 21 (B). Monocytes are CD14+/CD66b–, granulocytes are CD14–/CD66b+, megakaryocytes are CD41+/CD235a– and erythroid cells are CD41–/CD235a+. Representative scatter plots from an experiment are shown. Fractions of monocytes, granulocytes, megakaryocytes, and erythroid cells in the CD34– population on day 1 and day 21 are represented (C). Data presented are averages with standard error from at least three independent experiments. This figure has been modified from Bapat et al.11. Please click here to view a larger version of this figure.
Figure 4: Representative images of HSPCs and differentiated cells. CD34+ HSPCs (A) and cells from myeloid cultures at day 21 (B) were stained with the Wright-Giemsa stain. Cells corresponding to granulocytes (black arrows), monocytes (blue arrows) and erythroid cells (red arrows) are shown. Scale bars represent 10 μm. This figure has been modified from Bapat et al.11. Please click here to view a larger version of this figure.
The protocol described here is suitable for ex vivo differentiation of UCB derived CD34+ HSPCs to the four myeloid lineages. Initial incubation with a cytokine mix consisting of SCF, TPO, Flt3L and IL3 stimulates the CD34+ cells. Subsequently, differentiation is achieved with a cocktail of SCF, IL3, Flt3L, EPO, and TPO. In this mix, SCF, IL3, and Flt3L are important for survival and proliferation of CD34+ HSCs. EPO and TPO promote differentiation toward erythrocytes and megakaryocytes, respectively, and IL3 promotes differentiation of early granulocyte-monocyte precursors and mature cells13,17,18. One caveat to this method is the observed variation in the percentages of differentiated cells. This could be because of differences in capacities of the CD34+ HSPCs isolated from different donors to give rise to the lineage cells.
The layer of MS-5 cells is critical for efficient differentiation of the CD34+ HSPCs. In our experience, the MS-5 cells need to be plated at least 24 hours prior to seeding of the CD34+ cells. It is important to maintain the confluence of MS-5 cells at 80-90%. Overconfluent MS-5 cells tend to detach, and lower confluency causes reduced differentiation. During incubation, the CD34+ cells expand by ~50-fold. They become confluent by day 14 and need to be harvested and split into additional wells containing MS-5 cells on a new plate.
Frequency of media changes is also critical for efficient differentiation and must be performed every 3-4 days to maintain optimal cytokine concentrations. Fewer media changes and lower cytokine concentrations, both cause a reduction in proliferation and differentiation of CD34+ HSPCs. This requirement for large amounts of cytokines can significantly increase costs and thus, is a limitation of this protocol. Another limitation for large scale experiments is the number of CD34+ cells obtained from a unit of cord blood. Typically, a fresh UCB unit of ~100 mL yields about one million CD34+ cells. Storage of the unit beyond 24 hours significantly reduces the yield. Further loss of purity of the CD34+ cells can occur during the column wash steps that are important for removing any unlabeled contaminating cells. If the column clogs during the wash steps, it is recommended that the bound cells from the clogged column be eluted and reloaded on a new column in order to get a pure population of CD34+ cells.
In the literature, different cytokine combinations have been reported that promote differentiation towards either megakaryocytic, erythroid, or granulo-monocyte lineages19,20,21,22. An advantage of this protocol is that it allows differentiation along all four myeloid populations, namely monocytes, granulocytes, megakaryocytes, and erythrocytes. Thus, it can be employed for studying normal myeloid differentiation and for investigating the impact of myeloid disease (myelodysplastic syndromes, acute myeloid leukemia, myeloproliferative neoplasms, and others) associated point mutations and chromosomal translocations on molecular and cellular phenotype during proliferation and differentiation of CD34+ cells11,12,23,24,25. This protocol can also be employed for examining the impact of anti- and pro-inflammatory cytokines, and of potential therapeutic drugs on myeloid differentiation26,27,28.
The authors have nothing to disclose.
The authors would like to thank Wendy Barrett, Rachel Caballero, and Gabriella Ruiz of Maricopa Integrated Health Systems for the de-identified and donated cord blood units, Mrinalini Kala for assistance with flow cytometry, and Gay Crooks and Christopher Seet for advice on ex vivo myeloid differentiation. This work was supported by funds to S.S. from the National Institutes of Health (R21CA170786 and R01GM127464) and the American Cancer Society (the Institutional Research Grant 74-001-34-IRG). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
0.4% Trypan blue solution | Thermo Fisher Scientific | 15250-061 | Dilute working stock to 0.2% in sterile 1x PBS |
0.5 M UltraPure Ethylene diamine tetra acetic acid, pH 8.0 | Gibco | 15575-038 | |
10x Hanks Balanced Salt Solution (HBSS) | Invitrogen | 14185052 | Dilute to 1x with sterile distilled water & pH to 7.2 |
2.5% Trypsin, no phenol red | Thermo Fisher Scientific | 15090046 | Dilute working stock to 1x with sterile 1x PBS |
30 µm Pre-separation filters | Miltenyi biotech | 130-041-407 | |
35% sterile Bovine serum albumin | Sigma-Aldrich | A7979 | |
7-AAD | Biolegend | 420404 | Used as a live/dead stain to eliminate dead cells from FACS analysis |
Anti-human CD10-FITC antibody (Clone HI10a) | Biolegend | 312207 | Use 1:20 dilution |
Anti-human CD11b-FITC (activated) antibody (Clone CBRM1/5) | Biolegend | 301403 | Use 1:5 dilution |
Anti-human CD123-APC antibody (Clone 6H6) | Biolegend | 306012 | Use 1:20 dilution |
Anti-human CD14-PE antibody (Clone M5E2) | Biolegend | 301806 | Use 1:20 dilution |
Anti-human CD19-FITC antibody (Clone 4G7) | BD Biosciences | 347543 | Use 1:5 dilution |
Anti-human CD235a-APC antibody (Clone GA-R2 (HIR2)) | BD Biosciences | 551336 | Use 1:20 dilution |
Anti-human CD235a-FITC antibody (Clone HIR2) | Biolegend | 306609 | Use 1:50 dilution |
Anti-human CD34-APC-Cy7 antibody (Clone 581) | Biolegend | 343514 | Use 1:20 dilution |
Anti-human CD38-PE antibody (Clone HIT2) | Biolegend | 303506 | Use 1:20 dilution |
Anti-human CD3-FITC antibody (Clone UCHT1) | Biolegend | 300405 | Use 1:20 dilution |
Anti-human CD41a-PerCP-Cy5.5 antibody (Clone HIP8) | Biolegend | 303720 | Use 1:20 dilution |
Anti-human CD45Ra-PE-Cy7 antibody (Clone HI100) | Biolegend | 304126 | Use 1:20 dilution |
Anti-human CD66b-PE-Cy7 antibody (Clone G10F5) | Biolegend | 305116 | Use 1:20 dilution |
Anti-human CD7-FITC antibody (Clone CD7-6B7) | Biolegend | 343103 | Use 1:20 dilution |
Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231-100 | Filter sterilize before use |
Dulbecco’s Modified Eagle Medium (DMEM) powder with L-Glutamine | Gibco | 12100046 | Reconstitute 1 packet to make 1 L of DMEM media with sodium bicarbonate, 10% FBS & 1% penicillin & streptomycin |
Fetal bovine serum, Australian source, heat inactivated | Omega Scientific | FB-22 Lot #609716 | |
Human CD34 microbead kit | Miltenyi biotech | 130-046-702 | |
Human Thrombopoietin (TPO), research grade | Miltenyi biotech | 130-094-011 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 50 ng/mL for both myeloid differentiation & stimulation medium |
L-Glutamine | Omega Scientific | GS-60 | 2 mM concentration in stimulation medium |
LS Columns | Miltenyi biotech | 130-042-401 | |
MACS Multi stand | Miltenyi biotech | 130-042-303 | |
MidiMACS magnetic separator | Miltenyi biotech | 130-042-302 | |
MNC fractionation media (Ficol-Paque PLUS) | GE Healthcare Biosciences | 17-1440-03 | |
MS-5 cells | Gift from the laboratory of Gay Crooks, UCLA | ||
Paraformaldehyde | Sigma-Aldrich | P6148 | Heat 800 mL of 1x PBS in a glass beaker on a stir plate in a chemical hood to ~65 °C. Add 10 g of paraformaldehyde powder. To completely dissolve the paraformaldehyde, raise the pH by adding 1 N NaOH. Cool and filter the solution and make up the volume to 1 L with 1x PBS. Adjust the pH to 7.2. |
Penicillin & Streptomycin | Sigma-Aldrich | P4458-100ml | |
Poly-L lysine | Sigma-Aldrich | P2636 | Make a 10 mg/mL stock in 1x PBS |
Recombinant human erythropoietin-alpha (rHu EPO-α) | BioBasic | RC213-15 | Make a stock of 2000 units/mL in 1x PBS + 0.1% BSA. Use 4 units/mL for myeloid differentiation |
Recombinant human fibronectin fragment (RetroNectin) | Takara | T100B | Use 20 µg/mL diluted in sterile 1x PBS to coat wells prior to stimulation of CD34+ HSCs. |
Recombinant human Flt-3 ligand (rHu Flt-3L) | BioBasic | RC214-16 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 50 ng/mL in stimulation medium |
Recombinant human interleukin-3 (rHu IL-3) | BioBasic | RC212-14 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 20 ng/mL in stimulation medium |
Recombinant human stem cell factor (rHu SCF) | BioBasic | RC213-12 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 50 ng/mL in stimulation medium |
Serum free medium (X-Vivo-15) | Lonza | 04-418Q | |
Sodium bicarbonate | Fisher Scientific | BP328-500 | |
Wright-Giemsa stain, modified | Sigma-Aldrich | WG16-500 | Use according to manufacturer's instructions |
Equipment | |||
BD LSR II flow cytometer | BD Biosciences | ||
Centrifuge | Sorvall Legend RT | ||
Light microscope | Olympus |