scholarly journals Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells at Single Cell Resolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1187-1187
Author(s):  
Kim Vanuytsel ◽  
Carlos Villacorta-Martin ◽  
Wilfredo Garcia Beltran ◽  
Taylor Matte ◽  
Alejandro Balazs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Single Cell ◽  
Protein Level ◽  
Fetal Liver ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Surface Marker Expression

Intro: In the mouse, hematopoietic stem cells (HSCs) can be isolated and characterized at single cell resolution using a well-defined panel of markers. While it is possible to enrich for human HSCs using a panel of associated markers, similar resolution has not been attained. By profiling HSCs residing in the human fetal liver (FL) using a novel technique called CITE-Seq that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies, we aimed to establish an accurate molecular signature of engraftable human HSCs shortly after they arise in development. As HSCs are defined functionally, we have coupled this transcriptomic and protein-level characterization with transplantation assays in immunocompromised NOD scid gamma (NSG) mice to connect expression profiles of cell subsets with functional engraftment. Methods: CITE-Seq was performed on human FL cells (week 19) that showed robust engraftment capability in NSG mice. CD34+ and CD34- cells were magnetically separated and stained with a panel of 19 oligo-tagged antibodies that were deemed relevant to characterize HSCs, including classical HSC markers but also novel targets that were identified in a previous pilot scRNAseq experiment conducted on CD34+ FL cells. From the CD34+ fraction, we sorted live-gated cells (CD34+bulk) as well as a population of cells that was further enriched based on the expression of GPI-80, a marker tightly linked to engraftment potential (CD34+GPI-80+, ~3%). CD34-GlycophorinA(GYPA)- cells were also sorted to assay for the presence of CD34- HSCs. These fractions were then loaded onto the 10x Genomics platform for capture of single cells and subsequent reverse transcription and amplification of both mRNAs and antibody-derived tags (ADTs). Results: Both mRNA and ADT libraries were successfully sequenced, yielding 29-43,000 reads/cell for the mRNA portion and >1,500 reads/cell for the ADT fraction. After quality control and filtering, this effort resulted in 8,775 CD34+bulk cells, 7,279 CD34+GPI-80+ cells, and 6,937 CD34-GYPA- cells available for further analysis. Simultaneous transplantation experiments of the fractions assayed by CITE-seq revealed superior engraftment potential of the CD34+GPI-80+ fraction, confirming enrichment for bona fide HSCs at the functional level. This was also reflected in the scRNAseq data where we found enrichment for known HSC markers such as VNN2 (GPI-80), PROM1 (CD133), PROCR (EPCR), THY1 (CD90), ITGA6 (CD49f), HMGA2, CLEC9A and HLF in the CD34+GPI-80+ fraction compared to CD34+bulk cells. As our pilot studies revealed considerable differences in transcriptional expression (via scRNAseq) as compared to protein-level expression (via cell surface marker expression), integration of the transcriptomic and cell surface marker expression data will further refine the signature of engraftable HSCs. Both layers of information at single cell resolution will allow for the identification of novel markers or unique combinations of markers that are directly correlated with engraftment potential. Conclusion: By isolating the GPI-80+ population within the CD34+ fraction in human FL, we have achieved unprecedented resolution of the signature of engraftable HSCs as confirmed by transplantation experiments. The in-depth characterization of this compartment as well as the surrounding CD34+ and CD34- cells within the FL is expected to yield valuable insights with respect to several biological questions. This data can be directly harnessed in improving the purification and expansion of engraftable HSCs as well as in guiding the in vitro generation of HSCs from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

2012 ◽  
Vol 2 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Stefan Wirths ◽  
Elke Malenke ◽  
Torsten Kluba ◽  
Simone Rieger ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Surface ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Cell Surface Marker Expression

scholarly journals Immunophenotypic- and Molecular Analysis of Human Hematopoietic Stem and Progenitor Heterogeneity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3701-3701
Author(s):  
Mikael Sommarin ◽  
Parashar Dhapola ◽  
Linda Geironson Ulfsson ◽  
Fatemeh Safi ◽  
Eva Erlandsson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cell ◽  
Molecular Analysis ◽  
Surface Marker ◽  
Molecular Signature ◽  
Molecular Heterogeneity ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Surface Marker Expression ◽  
Prospective Isolation

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells lack stem cell activity, confounding molecular analysis of HSC function. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in single-cell CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of immunophenotype and RNA sequencing. This allowed us to correlate the molecular signature of each single-cell with the expression of 40 cell-surface proteins in CD34+ and CD34+CD38- populations of fetal liver (FL), CB, young- and old BM. Following sequencing, the cells were clustered based on molecular signature. Fourteen distinct groups with HSC-, multipotent progenitor-, and early committed progenitor profiles were identified. To investigate how the molecularly defined groups correlate to established populations within CD34+ HSPCs, the surface marker expression from the CITE-seq experiment was included in the analysis. The immunophenotypically defined GMP, MEP and CMP populations showed high molecular heterogeneity with cells at different stages of differentiation. The immunophenotypic HSCs (CD38-CD90+CD45RA-) correlated with the molecularly defined HSC population with a 75.6% overlap. To find novel surface markers for prospective isolation of HSCs pseudo-time analysis was used, allowing for correlation of surface marker expression with differentiation status. Interestingly, both CD35 and CD11a correlated with differentiation, with CD35 expression decreasing and CD11a expression increasing with pseudo-time. These two novel HSC marker-candidates are currently being functionally validated by transplantation analysis. To compare the progenitor composition of CD34+ HSPCs at different stages of life, young BM was used as a baseline control. Interestingly, compared to young BM CB CD34+ cells contained a higher frequency of multipotent progenitor cells and a decreased proportion of committed progenitors. In contrast, old CD34+ BM was reduced in multipotent progenitor frequencies with a corresponding relative increase of committed progenitors. However, both CB and old BM showed similar proportions of molecularly defined HSCs as compared to young BM. These results indicate that ageing causes a depletion of the earliest hematopoietic progenitor populations while the HSC pool remains intact. Together, using single cell CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify two novel cell-surface marker candidates for prospective isolation of HSCs. Disclosures No relevant conflicts of interest to declare.

3139 – TRANSCRIPTIONAL, PROTEIN-LEVEL AND FUNCTIONAL PROFILING OF HUMAN FETAL LIVER-DERIVED HEMATOPOIETIC STEM CELLS AT SINGLE CELL RESOLUTION

2020 ◽  
Vol 88 ◽  
pp. S81
Author(s):  
Kim Vanuytsel ◽  
Carlos Villacorta Martin ◽  
Jonathan Lindstrom-Vautrin ◽  
Zhe Wang ◽  
Wilfredo Garcia Beltran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Protein Level ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Human Fetal Liver ◽  
Functional Profiling

Comparative analysis of mesenchymal stem cell surface marker expression for human dental mesenchymal stem cells

2013 ◽  
Vol 8 (4) ◽  
pp. 453-466 ◽  
Author(s):  
Joo-Young Park ◽  
Ho-Jin Jeon ◽  
Tae Yun Kim ◽  
Kyeong-Yeoll Lee ◽  
Kyoungsook Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Comparative Analysis ◽  
Mesenchymal Stem Cell ◽  
Cell Surface ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Surface Marker Expression ◽  
Cell Surface Marker Expression

scholarly journals Multi-Modal Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells Reveals the Molecular Signature of Engraftment Potential

2020 ◽  
Author(s):  
Kim Vanuytsel ◽  
Carlos Villacorta-Martin ◽  
Jonathan Lindstrom-Vautrin ◽  
Zhe Wang ◽  
Wilfredo F. Garcia-Beltran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Biological Function ◽  
Fetal Liver ◽  
Surface Marker ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Surface Marker Expression ◽  
Cell Surface Marker Expression ◽  
Potential Integration

SUMMARYThe human hematopoietic stem cell (HSC) harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, HSCs in the fetal liver (FL) undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. We profiled 26,407 FL cells at both transcriptional and protein levels including over 7,000 highly enriched and functional FL HSCs to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression revealed a generalizable signature defining functional FL HSCs and allowed for the stratification of enrichment strategies with high translational potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers, serving as a useful resource for the field.

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