Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding

Verovskaya, Evgenia; Broekhuis, Mathilde; Zwart, Erik; Ritsema, Martha; Os, Ronald van; Haan, Gerald de; Bystrykh, Leonid

doi:10.1182/blood-2013-01-481135

Cited by 159 publications

(109 citation statements)

References 28 publications

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“…For example, a previous analysis of a similarly configured barcode identified 30–50 barcodes per transplanted mouse, which was sufficient to provide insights into hematopoietic differentiation (35). Other gene marking studies utilizing different barcode designs have detected fewer than 100 different barcodes in different lineages of transplanted mice (31,32). The reliability of such analyses could be further enhanced if fully characterized barcode libraries of moderate complexity, such as that in the present study, are used to validate data analysis strategies.…”

Section: Discussionmentioning

confidence: 99%

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

Deakin

Ginn

et al. 2014

Nucleic Acids Research

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Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved.

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Section: Discussionmentioning

confidence: 99%

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

Deakin

Ginn

et al. 2014

Nucleic Acids Research

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“…However, paradigm-shifting research on HSC clonality and the heterogeneous contribution of HSC clones to hematopoiesis has recently challenged this view. Higher levels of clonality, where only a few clones actively contribute to the production of peripheral blood cells, has been observed with hematopoietic aging [61-63]. Historically, an aging-related progression towards hematopoietic clonality is demonstrated by skewing towards individual X-chromosome inactivation associated with myeloid bias in aged female individuals [64, 65].…”

Section: Hsc Clonal Contribution To Hematopoietic Agingmentioning

confidence: 99%

“…They further imply, based on sophisticated mathematical models, that the aging-associated myeloid bias might be a consequence of reduced multipotent progenitor (MPP) flux to common lymphoid progenitors (CLP) rather than a change in the HSC pool composition [63]. Verovskaya et al, using cellular barcoding and high-throughput sequencing to monitor clonal contribution to regenerative hematopoiesis, concluded that young mice present fewer active HSC clones producing high numbers of progeny, while in aged mice more active HSC clones are observed, but which produce fewer progeny [61]. These findings appear contradictory, but one explanation might be that clonality in steady-state hematopoiesis might differ from clonality in a regenerative setting such as BM transplantation.…”

Section: Hsc Clonal Contribution To Hematopoietic Agingmentioning

confidence: 99%

Aging, Clonality, and Rejuvenation of Hematopoietic Stem Cells

Akunuru

Geiger

2016

Trends in Molecular Medicine

148

120

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Aging is associated with reduced organ function and increased disease incidence. Hematopoietic stem cell (HSC) aging driven by both cell intrinsic and extrinsic factors is linked to impaired HSC self-renewal and regeneration, aging-associated immune remodeling, and increased leukemia incidence. Compromised DNA damage responses and increased production of reactive oxygen species have been previously causatively attributed to HSC aging. However, recent paradigm-shifting concepts such as global epigenetic and cytoskeletal polarity shifts, cellular senescence, as well as clonal selection of HSCs upon aging provide new insights into HSC aging mechanisms. Rejuvenating agents that can reprogram the epigenetic status of aged HSCs or senolytic drugs that selectively deplete senescent cells provide promising translational avenues for attenuating hematopoietic aging and potentially, alleviating aging-associated immune remodeling and myeloid malignancies.

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“…Unlike the extensive polyclonal repopulation seen in primates, murine studies demonstrated the behaviors of a single or a few repopulating clones per mouse(Copley et al, 2012; Jordan and Lemischka, 1990; Lemischka et al, 1986; Muller-Sieburg et al, 2012; Smith et al, 1991). Some recent studies have investigated oligoclonal repopulation (dozens of clones) in mice (Cornils et al, 2012; Gerrits et al, 2010; Lu et al, 2011; Naik et al, 2013; Verovskaya et al, 2013), nevertheless, translating these studies into understanding human repopulation is limited because of the far greater complexity of polyclonal reconstitution as well as the greater demands placed on stem cells within the larger and longer-lived human system. Thus, despite the significant advances in our understanding of the HSC biology from murine models, the functional properties of primate HSPCs following transplant remain poorly characterized.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of HSPC Repopulation in Nonhuman Primates Revealed by a Decade-Long Clonal-Tracking Study

et al. 2014

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SUMMARY In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.

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Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding

Abstract: Key Points Quantitative clonal analysis demonstrates directional changes in contributions of stem cells to blood. The pool of aged hematopoietic stem cells is comprised of many, but small clones, while young stem cells are less numerous, but more potent.

Cited by 159 publications

References 28 publications

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

Aging, Clonality, and Rejuvenation of Hematopoietic Stem Cells

Dynamics of HSPC Repopulation in Nonhuman Primates Revealed by a Decade-Long Clonal-Tracking Study

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