Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential

Knapp, David J.H.F.; Hammond, Colin A.; Hui, Tony; Loenhout, Marijn T J van; Wang, Fangwu; Aghaeepour, Nima; Miller, Paul H.; Moksa, Michelle; Rabu, Gabrielle; Beer, Philip; Pellacani, Davide; Humphries, R. Keith; Hansen, Carl L.; Hirst, Martin; Eaves, Connie J.

doi:10.1038/s41556-018-0104-5

Cited by 49 publications

(42 citation statements)

References 44 publications

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“…Unlike recent studies on human HSC populations 2 , 49 , 50 , we administered human EPO to NSG mice to overcome the limitation in erythroid differentiation seen in this xenograft model 51 . We demonstrate that extending the range of lineage read-out to Ery differentiation significantly expands our understanding of heterogeneity within the phenotypic HSC/MPP pool, and identify rare multipotent (Ery/My/Ly) long-term repopulating HSC within the CLEC9A hi CD34 lo fraction of the HSC/MPP and 49f + HSC pools (Subset1).…”

Section: Discussionmentioning

confidence: 99%

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors

et al. 2018

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Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19−CD34+CD38−CD45RA−CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f+ compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.

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Section: Discussionmentioning

confidence: 99%

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors

et al. 2018

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“…22 Barcoding analyses of human CD34 1 HSPCs engrafted in nonobese diabetic-severe combined immunodeficiency gammaC 2/2 mice also suggest that the HSPC potential is heterogeneous in humans. 23,24 However, the long-term repopulation capacity is limited by the animal's life span, and the interpretation of these data in mice is complicated by a skewing of human cell differentiation toward lymphoid lineages.…”

Section: Introductionmentioning

confidence: 99%

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs

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Guilloux

Denis

et al. 2020

Blood

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In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

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“…These cells also possessed molecular characteristics consistent with being more deeply quiescent. Thus, these and other studies again point to the heterogeneity of human HSCs and the need for more defined biomarker identification of the most primitive HSCs …”

Section: Discussionmentioning

confidence: 82%

“…Although we have used hCD33 as a marker of the myeloid lineage after transplant, recent studies have identified this biomarker on a subset of human UCB hCD34 + CD38‐CD45RA‐CD90 + CD49f + HSCs, particularly on those that contain all the serial regenerative HSC activity in human UCB. These cells also possessed molecular characteristics consistent with being more deeply quiescent.…”

Section: Discussionmentioning

confidence: 99%

A modified CD34+ hematopoietic stem and progenitor cell isolation strategy from cryopreserved human umbilical cord blood

et al. 2019

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BACKGROUND: Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable. STUDY DESIGN:This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magneticactivated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice. RESULTS:Total nucleated cell recovery after thawing and washing was 44.7 AE 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 AE 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 AE 10.7% and 87.8 AE 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment.CONCLUSION: This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing. ABBREVIATIONS: BMRs = batch manufacturing records; CFUs = colony-forming units; cGMP = current good manufacturing practice; HSC = hematopoietic stem cell; HSCT = hematopoietic stem cell transplantation; HSPCs = hematopoietic stem and progenitor cells; LMPP = lymphoid-primed multipotent; MPP = Multipotent progenitors; SOPs = standard operating procedures; SRCs = severe combined immunodeficiency repopulating cells; TNCs = total nucleated cells; UCB = umbilical cord blood.

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Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential

Cited by 49 publications

References 44 publications

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs

A modified CD34+ hematopoietic stem and progenitor cell isolation strategy from cryopreserved human umbilical cord blood

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