Isolation of human VSELs and HSCs
Human umbilical cord blood (hUCB) was obtained from healthy newborns delivered at the Department of Obstetrics and Gynecology, Medical University of Warsaw (Warsaw Bioethics Committee permission number KB/3/2018). hUCB units, containing a minimum of 20 ml, were diluted with phosphate-buffered saline (PBS) and carefully layered over Ficoll-Paque (GE Healthcare, Chicago, IL, USA) and centrifuged for 30 min at 400x g at 4°C. The lymphocytes/monocytes/platelets phase was collected, washed, and used for further analysis. Briefly, cells were stained with the following antibodies: hematopoietic lineage markers (Lin) cocktail of antibodies, each FITC-conjugated: CD235a (clone GA-R2 [HIR2]), anti-CD2 (clone RPA-2.10), anti-CD3 (clone UCHT1), anti-CD14 (clone M5E2), anti-CD16 (clone 3G8), anti-CD19 (clone HIB19), anti-CD24 (clone ML5), anti-CD56 (clone NCAM16.2) and anti-CD66b (clone G10F5) (all BD Biosciences, San Jose, CA, USA); PE-Cy7-conjugated anti-CD45 (clone HI30, BioLegend, San Diego, CA, USA) and PE-conjugated anti-CD34 (clone 581, BioLegend, San Diego, CA, USA). Antibodies were used in the manufacturer's recommended concentration. Cells were stained in the dark at 4oC for 30 min, then centrifugated and resuspended in RPMI-1640 medium containing 2% fetal bovine serum (FBS, Corning Inc, Corning, NY, USA). Cells were sorted according to the strategy shown in Fig. 1A. Briefly, small events (4–12 µm in size) were included in the “lymphocyte-like” gate and then further analyzed for the expression of Lin marker, CD45, and CD34 antigens (Fig. 1). Populations of VSELs (Lin−CD45−CD34+) and HSCs (Lin−CD45+CD34+) were sorted on the MoFlo Astrios EQ cell sorter (Beckman Coulter, Brea, CA, USA).
Isolation of murine VSELs and HSCs
Animal studies were approved by the Animal Care and Use Committee of the Warsaw Medical University (Warsaw, Poland). Pathogen-free, 6–8-week-old C57BL/6 J wild-type (WT) mice were purchased from the Central Laboratory for Experimental Animals, Medical University of Warsaw. Bone marrow (BM) VSELs and HSCs were isolated from sacrificed WT mice. Briefly, cells were flushed from the tibias and femurs with cold PBS. Next, Red Blood Cells (RBCs) were lysed using 1× BD Pharm Lyse buffer (BD Pharmingen, San Jose, CA, USA). Obtained population of cells (MNCs + granulocytes) was washed and resuspended in RPMI-1640 medium (Corning Inc, Corning, NY, USA) containing 2% FBS (Corning Inc, Corning, NY, USA) and subsequently stained with the following anti-mouse antibodies: Lin antibodies cocktail, all PE, rat anti: CD45R/B220 (clone RA3-6B2), Gr-1 (clone RB6-8C5), TCRαβ (clone H57-597), TCRγδ (clone GL3), CD11b (clone M1/70), Ter119 (clone TER-119) (BD Pharmingen, San Jose, CA, USA); PE-Cy7 anti-CD45 (clone 30-F11) (BD Pharmingen, San Jose, CA, USA) and APC Ly-6A/E (Sca1) (clone E13-161.7) (BioLegend, San Diego, CA, USA). The cells were then washed and resuspended in RPMI-1640 medium plus 2% FBS, and the population of VSELs (Lin−Sca1+CD45−) and HSCs (Lin−Sca1+CD45+) were sorted using the MoFlo Astrios EQ cell sorter (Beckman Coulter, Brea, CA, USA) accordingly to strategy shown on the Fig. 1B.
Quantitative RT-PCR
Total RNA from murine and human VSELs, HSCs, and MNCs was isolated using RNeasy Micro and RNeasy Mini kit (Qiagen Inc., Hilden, Germany). RNA concentration was measured on NanoDrop, and at least 100 ng of RNA was reverse transcribed with the iScript reverse transcription kit (Bio-Rad, Hercules, CA, USA). The target genes were evaluated using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and specific primers listed in Tables 1 and 2. The samples were run on the CFX384 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). The PCR cycling conditions were 95°C (30s), 45 cycles at 95°C (15 s), and 60°C (30 s). A melting curve was created to emphasize the specificity of the primer and avoid the possibility of amplifying DNA contamination. Quantification was calculated using the comparative ΔCT method where mRNA levels of target receptors were normalized to the β-2microglobulin (human samples) or β-actin (murine samples) mRNA level. MNCs were assigned as a control group. According to melting point analysis, only one PCR product was amplified under these conditions. PCR products were visualized on 2% agarose gels.
Table 1
Sequenes of the human primers.
Human gene | Forward primer | Reverse primer |
β-2MG | TGGGTTTCATCCATCCGACA | TCAGTGGGGGTGAATTCA |
CD39 | TCGCCTCTATGGCAAGGACTAC | TCCAGGATGAAAGCATGGGTCC |
CD73 | AGTCCACTGGAGAGTTCCTGCA | TGAGAGGGTCATAACTGGGCAC |
A1 | TGCGAGTTCGAGAAGGTCATC | GAGCTGCTTGCGGATTAGGTA |
A2a | CGAGGGCTAAGGGCATCATTG | CTCCTTTGGCTGACCGCAGTT |
A2b | TAAAAGTTTGGTCACGGGGACCCGA | TTCACAAGGCAGCAGCTTTCATTCGT |
A3 | TACATCATTCGGAACAAACTC | GTCTTGAACTCCCGTCCATAA |
P2X1 | CCTCATCAGCAGTGTCTCTG | GGTCATGACCACGAAGGAGT |
P2X2 | GTGGAAATGAAAGACATCATCGTGCTGGT | AGGCCCAGGAGGAATCTGAATGGG |
P2X3 | GGTTTTCTTGCACGAGAAGGCTTACCA | TGAGGTGGCGTCACGTAATCAGACA |
P2X4 | CCTCTGCTTGCCCAGGTACTC | CCAGGAGATACGTTGTGCTCAA |
P2X5 | CTGCCTGTCGCTGTTCGA | GCAGGCCCACCTTCTTGTT |
P2X6 | AGGCCAGTGTGTGGTGTTCA | TCTCCACGGGGCACCAACTC |
P2X7 | AGTGCGAGTCCATTGTGGAG | CGCAGGTCTTGGGACTTCTT |
P2Y1 | GCCATCTGGATGTTCGTCTTCC | TGGCAGAGTCAGCACGTACAAG |
P2Y2 | CGAGGACTTCAAGTACGTGCTG | GTGGACGCATTCCAGGTCTTGA |
P2Y4 | TGCCTGGTCACTCTTGTTTG | GTACTCGGCAGTCAGCTTCC |
P2Y6 | AACCGCACTGTCTGCTATGACC | AGCAGGAAGCCGATGACAGTGA |
P2Y11 | AGGGCAAAGTGATGTTCCAC | CCCTCCAGGCTCTTCTTTCT |
P2Y12 | AACTGGGAACAGGACCACTG | ACATGAATGCCCAGATGACA |
P2Y13 | GCCGACTTGATAATGACACTCATG | CCTAACAGCACGATGCCCACAT |
P2Y14 | GCCGCAACATATTCAGCATCGTG | GCTGTAATGAGCTTCGGTCTGAC |
Table 2
Sequenes of the murine primers.
Murine gene | Forward primer | Reverse primer |
β-actin | ACCCCAGCCATGTACGTAGCCAT | CAGGATGGCGTGAGGGAGAGCAT |
CD39 | CTGGACAAGAGGAAGGTGCCTA | GACTGTCTGAGATGAGGCTTAGC |
CD73 | CGCTCAGAAAGTTCGAGGTGTG | CGCAGGCACTTCTTTGGAAGGT |
A1 | TTGTGGTAGGCCTGACACCCATGT | GCCGTTGGCTATCCAGGCTTGTT |
A2a | CGAAGGGCATCATTGCGATTTGCTG | ATGTAGGAAAAGACGATCATGTGCAAGACC |
A2b | TGCATTACAGACCCCCACCAACTACTTT | AAGAGGCTAAAGATGGAGCTCTGTGTGAG |
A3 | CTGGCCATTGCTGTAGACCG | GTCAGCCCCACCAGAAAGGA |
P2X1 | CCAGACCTCAAGTGGCCTTATCAGC | CTGGGAAGACATAGTCAGCCACGTC |
P2X2 | GGCGGTGTCATTGGGGTCATCAT | AAGAGGCAGGGTCATACTTGGGGT |
P2X3 | TCTCCAGCAGAGACATCAGCA | GGAGCATCTTGGTGAACTCAG |
P2X4 | CATTTATAATGCGCGGACGGATCCCT | CTCCACTGCCATCTCCTGAAAGCTG |
P2X5 | GGAAGATAATGTTGAGGTTGAG | TCCTGATGAACCCTCTCCAGT |
P2X6 | CCCAGAGCATCCTTCTGTTCC | GGCACCAGCTCCAGATCTCA |
P2X7 | CAGTATGAGACAAACAAAGTCACCCGGA | ATGTAGGAAAAGACGATCATGTGCAAGACC |
P2Y1 | CCTGCTATGACACCACGTCCAA | AGCGGAGAGTTGTCCAGGTCAT |
P2Y2 | TTCACCTGGCAGTTTCGGACTC | GTGTAGAAGAGGAAACGCACCAG |
P2Y4 | CTGGACAGTCATCTTCTCGGCT | TTCGGCGTTCAACAGTCTTGCC |
P2Y6 | CAGTCTTTGCTGCCACAGGCAT | AGCAAGAAGCCGATGACCGTGA |
P2Y10 | GAGCCAGAAACTGGAAGCGTAG | GGCTAAGCCAGCATTTCTCAGG |
P2Y12 | CATTGACCGCTACCTGAAGACC | GCCTCCTGTTGGTGAGAATCATG |
P2Y13 | TGGCATCAGGTGGTCAGTCACA | TTGTGCCTGCTGTCCTTACTCC |
P2Y14 | ACCTCCGTCAAGAGGAAGTCCA | GCTGTAGTGACCTTCCGTCTGA |
Isolation of Sca1+ cells from murine bone marrow
Pathogen-free, 6–8-week-old C57BL/6 J WT mice were purchased from the Central Laboratory for Experimental Animals, Medical University of Warsaw. Mice were sacrificed, and cells were flushed from the tibias and femurs with cold PBS. Next, RBCs were lysed using 1× BD Pharm Lyse buffer (BD Pharmingen, San Jose, CA, USA). Subsequently, BM cells were labeled with the Anti-Sca-1 MicroBead Kit (Vio® Bright FITC) (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Sca1+ cells were then isolated on a QuadroMacs separator with the use of LS columns (both Miltenyi Biotec and Bergisch Gladbach, Germany).
Isolation of CD34+ cells from human umbilical cord blood
hUCB was obtained from healthy newborns delivered at the Department of Obstetrics and Gynecology, Medical University of Warsaw. hUCB units, containing a minimum of 20 ml, were diluted with PBS and carefully layered over Ficall-Paque (GE Healthcare, Chicago, IL, USA) and centrifuged for 30 min at 400x g at 4°C. Phase containing MNCs was collected, and cells were then washed and labeled with anti-CD34 beads using a CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ cells were isolated on a QuadroMacs separator with the use of LS columns (both Miltenyi Biotec and Bergisch Gladbach, Germany).
Transwell migration assay
Cell migration was evaluated with the use of the Boyden Chamber system. Murine Sca1+ or human CD34+ cells were starved in RPMI-1640 medium containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MO, USA) for 1h or with 0.5% BSA RPMI-1640 containing 10 µM MCC950 (MedChemExpress, Princeton, NJ, USA). Lower chambers of a Costar Transwell 24-well plate (Corning Inc, Corning, NY, USA) (min. two wells per group) were pre-filled with 0.5% BSA RPMI-1640 for control or with 10 µM adenosine triphosphate (ATP, Sigma-Aldrich, Saint Louis, MO, USA) or 10 µM ATP with 10 µM Adenosine (Ado, Sigma-Aldrich, Saint Louis, MO, USA) or with 10 µM ATP and 10 µM MCC950. Cells were loaded onto the inserts (6.5 mm diameter membrane with 5 µm pores, Corning Inc, Corning, NY, USA) where at least 3x105 Sca1+ or 5x104 CD34+ cells were applied. The inserts were placed on a 24-well plate. The plate was incubated for 3 h at 37°C in a 5% CO2 incubator. Following incubation, medium from the lower chamber containing migrated cells was harvested and centrifuged. Cells were resuspended in 2% FBS RPMI-1640 and stained with the following antibodies: murine cells – Lin cocktail (described above), APC anti-Sca1 (clone E13-161.7) and V450 anti-CD45 (clone 30-F11) (BD, Biosciences, San Jose, CA, USA); human – Lin cocktail (described above), PE anti-CD45 (clone HI30, BD Biosciences, San Jose, CA, USA) and APC anti-CD34 (clone 581, BD Biosciences, San Jose, CA, USA). Samples were analyzed on FACS Verse (BD Biosciences, San Jose, CA, USA) where murine VSELs (Lin−Sca1+CD45−), HSCs (Lin−Sca1+CD45+), and human VSELs (Lin−CD34+CD45−) and HSCs (Lin−CD34+CD45+) were counted.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software Inc). All data are provided as an average ± SD. Statistical data analysis was performed using multiple unpaired t-tests with Welsh correction. In all calculations, p < 0.05 was considered significant.