The impact of aging on primate hematopoiesis as interrogated by clonal tracking

Abstract

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.

Aberrant Clonal Hematopoiesis Following Lentiviral Transduction of HSPC in a Rhesus Macaque

“…We developed a lentiviral barcoding model to quantitatively study the output of thousands of individual HSPC following autologous transplantation 15-18 . As part of our ongoing studies, we transplanted animal ZL34 with autologous CD34 + HSPC transduced with a lentiviral barcoding vector driving GFP expression via the strong murine retroviral MSCV promoter-enhancer inserted within the lentiviral long terminal repeat(LTR)(Figure 1A).…”

“…As part of our ongoing studies, we transplanted animal ZL34 with autologous CD34 + HSPC transduced with a lentiviral barcoding vector driving GFP expression via the strong murine retroviral MSCV promoter-enhancer inserted within the lentiviral long terminal repeat(LTR)(Figure 1A). The transduction/transplantation protocols utilized were unchanged from our prior experience 15-18 and the multiplicity of infection(MOI), CD34 + transplanted dose(cells/kg), and GFP% of the infused cells were within the ranges utilized for prior animals(Table S1, n = 10). ZL34, along with another monkey, ZL40(Table S1), received CMV-suppressing cidofovir for 4 months to study the impact of CMV reactivation on immune reconstitution.…”

“…CD34 + HSPC were collected from ZL34, a 4-year old male rhesus macaque, following mobilization with G-CSF/AMD1300, transduced(MOI=25) with a barcoded lentivirus library, and reinfused into the macaque following TBI as described 15-18 . ZL34 received weekly dosing with 5mg/kg cidofovir to suppress CMV reactivation.…”

“…Following gel purification, 15-24 multiplexed samples were used to create a DNA library for sequencing on an Illumina HiSeq2500/3000. Custom Python code(https://github.com/d93espinoza/barcode_extracter) was used for extraction and processing of barcodes from FASTQ files as described 15-18 . For modified barcode recovery, “Starcode” software(https://github.com/gui11aume/starcode) was used for extraction and processing of barcodes.…”

“…Our laboratory has used a rhesus macaque (RM) genetic barcoding model to study the output of lentivirally-transduced transplanted HSPC at a clonal level over time 15-18 ; such a setting closely resembles that of lentiviral HSPC gene therapy trials in which autologous HSPC are collected, transduced, and reinfused into the patient. In our published and unpublished studies, we have tracked over 101,000 individual lentiviral vector insertions in 9 transplanted macaques followed for up to six years and observed no clonal expansions associated with a transformed or aberrant hematologic phenotype.…”