C1 system

For single cell LSKFlt3⁻ gene expression analysis, cell capture and target preamplification were performed using the C1 system (Fluidigm) according to the manufacturer’s instructions. Real-time PCR was performed using the BioMark 96.96 Dynamic Array platform (Fluidigm) and DELTAgene assays (Fluidigm; Table S2). In total, >60 single cells from both Epo-exposed and control mice (∼20 cells from each of 3 biological replicates) were analyzed, and those scoring positive for Kit mRNA expression (>85% of cells analyzed) were included in the analysis. ΔCt values relative to Kit (Ct(Kit) − Ct(Gene)) were zero centered for each gene by subtraction of the mean value of all positive cells for the gene. These normalized values were used to generate heat maps, and to calculate the cumulative significance of Epo regulation of E and GM genes (i.e., all E genes without Epo against all E genes with Epo, and the same comparison for GM genes) using Student’s t test.

Single‐cell capture, lysis, and cDNA preparation were performed with the Fluidigm C1 system. Cells were loaded on a microfluidic C1 Single Cell Auto Prep Array for mRNA Seq (5–10 μm), and capture efficiency evaluated using microscopy. Lysis, reverse transcription, and cDNA amplification were performed with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech/Takara) according to Fluidigm's guidelines for single‐cell RNA‐seq on the C1 system. cDNA was harvested, profiles checked on the Fragment Analyzer (AATI), and their concentration determined using Quant‐iT PicoGreen dsDNA Assay Kit. For subsequent library preparation using Nextera XT DNA library preparation kit (Illumina) following the Fluidigm manual, cDNAs were normalized to 0.3 ng/μl. Libraries were pooled and sequenced SR75 on an Illumina NextSeq 500 system (75 cycles High Output v2 kit).

FACS‐isolated hiPSC‐derived EpCAM⁺ cells from FD‐AOs (FD‐EpCAM⁺ cells) or FF‐AOs (FF‐EpCAM⁺ cells) and 2D‐iAT1 cells were dissociated and prepared at a concentration of 400 cells/μL for single‐cell analyses. Single cells were loaded onto a 10‐ to 17‐μm‐diameter C1 Integrated Fluidic Circuit (#100‐6041; Fluidigm) for FD‐AOs and FF‐AOs and 17‐ to 25‐μm‐diameter C1 Integrated Fluidic Circuit (#100‐5761; Fluidigm) for 2D‐iAT1 cells with the C1 system (Fluidigm). A SMART‐Seq v4 Ultra Low Input RNA kit (635025; Takara Bio) was used for first‐strand cDNA synthesis and amplification in accordance with the manufacturer's instructions. Before first‐strand cDNA synthesis, RNA spike‐in controls (spikes 1, 4, and 7) (AM1780; Thermo Fisher Scientific) were added to cell lysates to evaluate the subsequent quantification according to the manufacturer's instructions. Sequencing libraries were prepared with a Nextera XT kit (Illumina). Sequencing was performed using an Illumina HiSeq2500 sequencer (Illumina). For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B). Sequencing was performed using an Illumina HiSeq3000 sequencer (Illumina). Bioinformatic analyses are described in the Materials and Methods in Supporting Information.

Passage 1 cells from human tendon were suspended, and single cells were captured on a microfluidic chip on the C1 system (Fluidigm). Amplified complementary DNA libraries were generated using the C1 Single-Cell Auto Prep Module 1 and Module 2 kits (Fluidigm) and a mixture of outer primers specific to 46 genes (table S1). Each gene was analyzed in duplicate. Genes were selected on the basis of established markers for multipotent stem cells and tendon lineage reported in the scientific literature, in addition to nestin and one reference gene (table S1). Single-cell qRT-PCR analyses were performed with inner (nested) primers on the BioMark platform (Fluidigm) using 96.96 Dynamic Array IFC chips (Fluidigm), according to the manufacturer’s instructions. HC, PCA, violin plots, ANOVA, and coexpressed gene identification were performed using the SINGuLAR Analysis Toolset 2.1. SPADE was performed on http://cytobank.org/. A background C_t of 28 was used.