Lymphoid Priming in Human Bone Marrow Begins Prior to CD10 Expression with Up-Regulation of L-selectin
ABSTRACT: Studies of adult human hematopoiesis have until now relied on the expression of CD10 to define lymphoid commitment. We report a novel lymphoid-primed population in human bone marrow that is generated from hematopoietic stem cells (HSC) prior to the onset of CD10 expression and B cell commitment, and is identified by high levels of the homing molecule L-selectin (CD62L). CD10-CD62Lhi progenitors have full lymphoid (B/T/NK) potential, and show reduced myeloid and absent erythroid potential. Genome-wide gene expression analysis demonstrates that the CD10-CD62Lhi population represents an intermediate stage of differentiation between CD34+CD38- HSC and CD34+lin-CD10+ progenitors marked by down-regulation of TAL1 and MPL, upregulation of E2A, CD3E and IL2RG expression, and absent B cell commitment or RAG1/2 expression. Immature CD34+CD1a- thymocytes are also CD62Lhi and L-selectin ligands are expressed at the cortico-medullary junction, suggesting a possible role for L-selectin in human thymic homing. These studies identify the earliest stage of lymphoid priming in human bone marrow. Freshly harvested human bone marrow was ficoll purified, enriched for CD34+, and then FACS sorted. It was then sorted into 3 samples CD34+CD38-, CD34+ CD10- CD62L++, and CD34+ CD10+, with 3 independent biological replicates
Project description:To elucidate the transcriptional ‘landscape’ that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNA (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus. Overall design: We performed RNA-Seq of 10 distinct cell types isolated by fluorescence activated cell sorting (FACS). From BM, we isolated CD34+CD38neglinneg cells, a population highly enriched for HSC, as well as three lymphoid progenitor populations; LMPP (CD34+CD45RA+CD38+CD10neg CD62Lhilinneg), CLP (CD34+CD38+CD10+CD45RA+linneg ) and fully B cell committed progenitors (BCP, CD34+CD38+CD19+). From thymus we isolated three CD34+ subsets; Thy1 (CD34+CD7neg CD1aneg CD4negCD8neg), Thy2 (CD34+CD7+CD1aneg CD4negCD8neg), and Thy 3 (CD34+CD7+CD1a+CD4negCD8neg), as well as fully T cell committed populations CD4+CD8+ (Thy 4), CD3+CD4+CD8neg (Thy5) and CD3+CD4neg CD8+ (Thy6).
Project description:Here we tracked transcriptome changes over ten human hematopoietic stem and early progenitor populations, defining the transcriptional dynamics underlying the first steps of commitment. Most of the transcriptional programs observed extended beyond lineage boundaries. In particular, multi-lymphoid progenitors (MLPs) presented a hybrid transcriptional state with elements of lymphoid and myeloid programs, but also stem cell characteristics. Total RNA was obtained from flow-sorted populations of human cord blood based on the cell surface expression of CD34, CD38, CD45RA, Thy1, and CD49f, CD10, CD7, CD19 and CD1a.
Project description:Cord blood (CB) samples from normal donors were obtained with informed consent. Fresh CB samples were processed within 18-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated. CB CD34+ enriched fraction was lineage depleted by staining with purified anti-human CD2, CD3, CD4, CD7, CD8a, CD11b, CD14, CD19, CD20, CD56, CD235a followed by Qdot 605 conjugated goat F(ab')2 anti-mouse IgG (H+L). Cells were also stained with anti-human CD38-FITC, CD45RA-PE or -BV650, CD123-PE Cy7, CD90-biotin, CD34- PerCP and CD10-APC. Finally, cells were incubated with streptavidin-conjugated APC-eF780 and Hoechst 33258 (Invitrogen, final concentration: 1 g/ml). Populations were defined, as follows: HSC - Lin-CD34+CD38-CD90+CD45RA-CD10-, MPP - Lin-CD34+CD38-CD90-CD45RA-CD10-, LMPP - Lin-CD34+CD38-CD90-/loCD45RA+CD10-, MLP - Lin-CD34+CD38-CD90-/loCD45RA+CD10+, GMP - Lin-CD34+CD38+CD123+CD45RA+CD10-, CMP - Lin-CD34+CD38+CD123+CD45RA-CD10-, MEP - Lin-CD34+CD38+CD123-CD45RA-CD10-.
Project description:We used whole genome transcriptome as gene discovery to dissect the developmental organization of human lymphopoiesis. Overall design: Gene expression of human CD127-CD7-/+ (CD34++CD45RA+ITGB7++CD127-CD2-CD115-CD116-CD123-CD10-CD19-CD24-) or CD127+CD7-/+ (CD34++CD45RA+ITGB7++CD127+CD2-CD115-CD116-CD123-CD10-CD19-CD24-) Early Lymphoid Progenitors (ELPs) as compared to Lymphoid-Mono-Dendritic Progenitors (LMDPs: CD34++ CD45RA+CD7-ITGB7-CD127-CD2-CD115-CD116-CD123-CD10-CD19-CD24-). Cells were extracted from the bone marrow of NSG mice engrafted with neonatal UCB CD34+ HPCs (NSG) or from of 21-week-old fetal donor (PRIM).
Project description:To characterize early human hematopoiesis on a single-cell level we developed an approach termed index-omics, which combines flow-cytometric, single-cell transcriptomic and single-cell lineage fate data. Healthy human bone marrow was labeled with a panel of up to 11 FACS surface markers commonly used to identify human hematopoietic stem and progenitor cells (HSPCs). Lin-CD34+38+ progenitors and Lin-CD34+CD38- stem cell enriched HSPCs were individually sorted, their surface marker fluorescence intensities recorded, and subjected to single-cell RNAseq or single-cell ex vivo cultures. Overall design: For transcriptomics, human bone marrow was stained with antibodies against Lineage markers (CD4, CD8, CD11b, CD14, CD19, CD20, CD56 and CD235a), CD7, CD10, CD34, CD38, CD45RA, CD49f, CD90 and CD135. For individual 1, 8 96-well plates of Lin-cd34+cd38+ and 6 96 well plates of Lin-cd34+cd38- cells were sorted into lysis buffer and subjected to a modified smart-seq2 protocol. For individual 2, 4 96-well plates of Lin-cd34+cd38+, 7 plates of Lin-cd34+cd38- and 1 plate of Lin-cd34+cd38-cd45RA-cd90+ were sorted into lysis buffer and subjected to QUARTZ Seq (Sasagawa et al 2013). For single-cell culture, human bone marrow from a third individual was stained with antibodies against CD2, CD34, CD38, CD45RA, CD71, CD90, CD130, CD135, FCER1A, KEL and a Lineage cocktail as above, plus CD10. Lineage output was then determined by automated FACS following prolonged ex-vivo cultivation. The files transcriptomics_raw_filtered_I1.csv and transcriptomics_raw_filtered_I2.csv contain the raw read counts for the cells that passed quality control. transcriptomics_normalized_filtered_I1.csv and transcriptomics_raw_filtered_I2.csv contain the readcounts normalized by posterior odds ratio (POR) as described in our manuscript. The files transcriptomics_facs_indeces_filtered_I1.csv and transcriptomics_facs_indeces_filtered_I2.csv contain the FACS surface marker expression for the cells that passed filter. Additionally, FACS surface marker expression is given as a characteristic of each sample. The file culture_data.csv contains the FACS surface marker expression, quantification of cell types in the mature colony, and scoring of colony type of the cells that were subjected to ex-vivo culture.
Project description:Gene expression profiles of CD34+CD38- stem cells and more differentiated CD34+CD38+ progenitor cells were compared. Comparison of expression profiles of hematopoietic stem cells from fetal liver, umbilical cord blood, bone marrow and mobilized pheripheral blood allowed us to identify a unique set of genes with conserved expression during ontogeny. Experiment Overall Design: CD34+CD38- en CD34+CD38+ cell populations were isolated by cell sorting from human Bone Marrow, mobilized peripheral blood, umbilical cord blood and fetal liver. Total RNA was isolated from each cell population followed by the synthesis of biotinylated cRNA. After fragmentation the biotinylated cRNA was hybridized to affymetrix U133A chips.
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations from patients with acute myeloid leukemia (AML) and a normal karyotype. We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients with normal karyotype and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).
Project description:Human embryonic stem cells (hESCs) offer an important model for investigating the human hematopoietic celldevelopment. Here, we used long serial analysis of gene expression and quantitative real-time PCR to characterize two subsets of primitive hematopoietic cells derived in vitro from hESCs. This revealed differences in their expression of genes associated with lymphoid and myeloid development, cellular biosynthetic processes, and cell cycle regulation. Further comparisons with analogous data for primitive hematopoietic cells isolated from first trimester human fetal liver and newborn cord blood showed a strong similarity between the transcriptomes of the most primitive hESC- and in vivo-derived populations, with the main differences involving genes that regulate HSC development, self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and non-coding RNAs. These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected. Overall design: Human embryonic stem cells (hESCs) are capable of indefinite self-renewal but can also be induced to undergo a stepwise process of differentiation into a spectrum of recognizable mature blood cell types. However, a clear understanding of the molecular mechanism by which the first hematopoietic stem cells (HSCs) acquire their unique defining properties of self-renewal and repopulating potential is lacking. As a first step towards obtaining the information needed to close this gap, we have undertaken a comparative gene expression analysis of different highly purified primitive human hematopoietic subpopulations (erythroid-megakaryocytic progenitor enriched CD43+CD235a+CD41a+/- cells, mutiplepotent progenitor enriched lin-CD34+CD43+CD45-, and lin-CD34+CD43+CD45+ cells) generated either in vitro from hESCs or in vivo from fetal (human fetal liver lin-CD34+CD38- cells) or neonatal hematopoietic primitive cells (human cord blood lin-CD34+CD38- and lin-CD34+CD38+ cells). This involved preparing a long serial analysis of gene expression (LongSAGE) library from an extracts of each prospectively isolated subpopulation and then sequencing each library to a depth of 200,000 tags.
Project description:Investigation of human hematopoietic stem cells gene expression patterns originating from different stages of ontogeny including fetal blood, cord blood, bone marrow, and mobilized peripheral blood in Lin-CD34+CD38- versus Lin-CD34+CD38+ populations. Experiment Overall Design: this experiment include 7 samples and 42 replicates