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Three dimension polypeptide scaffolds facilitates to expand rare circulating hematopoietic stem/progenitor cells in normal peripheral blood

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122682 | GEO | 2021/01/01

REPOSITORIES: GEO

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Project description:Autologous hematopoietic stem cell transplantation (ASCT) improves survival in multiple myeloma (MM). However, up to 30% of MM patients are unable to collect optimal numbers of peripheral blood (PB) CD34+ hematopoietic stem and progenitor cells (HSPCs) with standard G-CSF mobilization. Motixafortide is a novel cyclic-peptide CXCR4 inhibitor with extended receptor occupancy and enhanced in-vivo activity. The GENESIS Trial is a prospective, phase III, double-blind, placebo-controlled, international, multicenter, superiority trial with 122 adult patients randomized (2:1) to motixafortide+G-CSF or placebo+G-CSF for HSPC mobilization for ASCT in MM (NCT03246529). The primary endpoint of the study was met, with motixafortide+G-CSF enabling 92.5% of patients to collect ≥6x106 CD34+ cells/kg within 2 apheresis procedures versus 26.2% with placebo+G-CSF (p<0.0001). A key, prespecified secondary endpoint was also met, with motixafortide+G-CSF enabling 88.8% of patients to collect ≥6x106 CD34+ cells/kg in 1 apheresis versus 9.5% with placebo+G-CSF (p<0.0001). Motixafortide+G-CSF was safe and well-tolerated, with the most common treatment-emergent adverse events observed being transient, Grade 1/2 injection site reactions (pain: 50%, erythema: 27.5%, pruritis: 21.3%). In addition, extended immunophenotyping and single-cell transcriptional profiling were performed on CD34+ HSPCs mobilized on the GENESIS Trial, as well as a contemporaneous MM cohort undergoing plerixafor+G-CSF mobilization for ASCT and 3 cohorts of allogeneic HSPC donors undergoing single-agent mobilization with motixafortide, plerixafor or G-CSF alone. Motixafortide+G-CSF resulted in a 10.5-fold increase in primitive HSPCs collected versus placebo+G-CSF (p<0.0001); and significantly greater numbers of early stem and progenitors versus both placebo+G-CSF (p<0.0001) and plerixafor+G-CSF (p=0.0327). Motixafortide also preferentially mobilized HSPC subsets with upregulated gene expression pathways associated with enhanced self-renewal, regeneration and quiescence (EGF, NFB/TNF and HBO1). In conclusion, motixafortide+G-CSF mobilized significantly greater CD34+ HSPC numbers within 2 apheresis procedures versus placebo+G-CSF. Motixafortide also preferentially mobilized primitive stem and early progenitor cells with unique transcriptional profiles versus plerixafor and G-CSF.

Project description:Purpose: We designed this study to evaluate the feasibility of using only one factor to respecify human induced pluripotent stem cells (iPSCs)-derived blood cells into long-term engraftable hematopoietic stem and progenitor cells (HSPCs). We also parallelly compared iPSC-derived HSPCs (iHSPCs) with primary HSPCs under the same induction and transplantation condition in order to examine the functional equivalency between iHSPCs and bona fide HSPCs. Methods: In vitro derived human iPSC-HSPCs are induced with or without (w/o) doxycycline for the expression of MLL-AF4. In vivo derived bone marrow cells are harvested and FACS-sorted for human populations from primary transplants over nine to sixteen weeks after transplantation. Either iPSC-HSPCs or primary HSPCs, both of which are induced by MLL-AF4, is used for the transplantation experiments. iPSCs are either derived from peripheral blood mobilized CD34+ HSPCs (CD34-iPSC) or mononuclear cells (MN-iPSCs). Normal human CD34+ HSPCs and mononuclear cells are set as control groups. Results: MLL-AF4 can impart HSC and lymphoid potential to iPSC-derived blood cells in vitro, and induction of MLL-AF4 leads to the cellular identity transition from common myeloid progenitors to hematopoietic stem and progenitor cells. MLL-AF4 alone is sufficient to realize the potent engraftment of induced HSPCs (iHSPCs) from iPSCs, and multilineage and long-term hematopoiesis could be observed. Primary HSPCs with the induction of MLL-AF4 could gain significantly enhanced engraftability. During the long-term engraftment period, leukemic mutations with a bias to B-cell leukemia could be found in the iHSPCs and their derivatives. By contrast, MLL-AF4 induced primary HSPCs maintain the normal hematopoiesis without leukemic transformation. Conclusions: Our study has demonstrated for the first time, to our knowledge, that the pluripotency-dependent conversion of somatic cells to long-term engraftable HSPCs can be achieved by using a single factor in a non-integrative way. This study also suggests that iPSC-derived HSPCs are more prone to leukemic mutations during the long-term engraftment period, which provides a necessary caveat of using them in the actual therapies.

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Three dimension polypeptide scaffolds facilitates to expand rare circulating hematopoietic stem/progenitor cells in normal peripheral blood

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