GSE122682
Three dimension polypeptide scaffolds facilitates to expand rare circulating hematopoietic stem/progenitor cells in normal peripheral blood
Purpose: Circulating hematopoietic stem/progenitor cells (cHSPCs) are rare in normal human peripheral blood (PB) without mobilization. We apply a novel three-dimension culture system (3DCS) seeded with no mobilized PB monocytes (PBMNCs), to expand cHSPCs. Two-dimension culture system (2DCS), primary PBMNCs and CD34+ HSPCs derived from bone marrow serves as system, negative and positive control groups respectively. Bulk RNA sequencing analysis is performed to dissect the function and molecular mechanism for cHSPCs. Methods: Four groups are designed in the study including the samples cultured in 2DCS, cultured in 3DCS, normal primary PBMNCs and CD34+ HSPCs derived from mobilized peripheral blood. Total RNA was extracted from the cells followed by mRNA enrichment with poly-T oligo-attached magnetic beads. Cells from 2DCS and normal primary PBMNCs served as controls (the number of repetitions for the groups, n = 3). Mobilized bone marrow-derived CD34+ HSPCs were purified with EasySep Human CD34 Positive Selection Kit (StemCell Technologies) serving as positive control (n = 4). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit (NEB, Illumina®, USA) following manufacturer's recommendations. Index codes were added to attribute sequences to each sample. Preferential 150~200 bp in length of cDNA fragments were selected and purified with AMPure XP system (Beckman Coulter, Beverly, USA). PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and index (X) Primer after adaptor of cDNA being ligated with USER Enzyme (NEB, USA). At last, PCR products were purified with AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded products was carried out on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. Sequencing raw data were generated using the Illumina HiSeqTM platform, and 125 bp/150 bp paired-end reads were selected. Results: Bulk RNA sequencing analysis reveals that the samples in 3DCS have more similarity expression pattern with CD34+ hematopoietic stem/progenitor cells (HSPCs) than ones in 2DCS and PBMNCs, for their expression higher level of the specific hematopoietic stem signatures including core stem transcription factors (TFs) as Runx1, HoxA9, HoxB4 and GATA2, self-renewal genes, surface epitopes, and some signaling pathways essential for HSPC fate determination. This study provides a novel culture system to expand rare cHSPC in normal PB, and the underlying molecular and functional signatures are dissected. Conclusions: Our study demonstrates the first detailed analysis of normal peripheral blood-derived cHSPC transcriptomes based on RNA-seq technology. Our results show that 3DCS facilitates to expand the rare circulating HSPCs with repopulating capacity. The underlying molecular and functional signatures of cHSPCs are dissected. The study provides a platform for normal PB further clinical applications in cell transplantation and personalized precision medicine. Overall design: mRNA profiling by high throughput sequencing to dissect the function and molecular mechanism for 4 different groups including the samples from 2DCS, 3DCS, primary normal PBMNCs and CD34+ HSPCs purified from mobilized peripheral blood.