The Impact of Clinically Relevant Culture on Human Haematopoietic Stem Cells: A Kinetic Analysis at Single Cell Resolution

Haematopoietic stem cell (HSC) ex vivo gene therapy is now successfully used to treat an in-creasing number of monogenic disorders affecting the blood system. The ultimate efficacy of this therapy depends upon successful targeting of long-term haematopoietic stem cells (LT-HSCs), which are characterised by a predominantly quiescent status and sustained self-renewal capacity. Clinical ex vivo gene therapy protocols target the heterogenous mix of stem and pro-genitor cells encompassed in the CD34+ fraction with a limited understanding of how ex vivo manipulation impacts a purified LT-HSC subset. Importantly, long-term repopulation capacity is lost over culture, although the kinetics and molecular drivers of this process remain unclear. To address these questions, I perform single cell RNA-Seq, in vitro functional assays and in vivo transplantation in a time resolved manner following cord blood (CB) culture in differentiation facilitating conditions and mobilized peripheral blood (mPB) culture during a lentiviral ex vivo gene therapy protocol. First, I characterise the molecular impact of a 62 hr lentiviral ex vivo gene therapy protocol on mPB LT-HSC, short-term HSC (ST-HSC) and CD34+ cells. I reveal that the ex vivo protocol dramatically rewires the quiescent LT-HSC transcriptome and demonstrate that the majority of transcriptional changes are attributed to the culture conditions rather than lentiviral transduction. Secondly, I refine the kinetics associated with HSC functional attrition over the first complete cell cycle ex vivo. Long-term repopulation capacity is maintained for the first 6 hr irrespective of HSC source or tested culture conditions. I identify the 6 hr time-point as encompassing an adaptation period where LT-HSCs are rapidly responding to the instructive signals of culture and is molecularly underpinned by transient upregulation of cell stress response signalling. Follow-ing the 6 hr time-point, long-term repopulation capacity drops dramatically by 24 hr in CB culture and 62 hr in mPB culture. Loss of HSC function is correlated with reduced survival, cell cycle progression (late G1-M), sharp upregulation of MYC and a reduced ability to resolve proteostatic stress. Taken together, my results demonstrate that the 6 to 24 hr transition point is instrumental for the determination of LT-HSC cell fate ex vivo. Finally, using an ex vivo culture system of reversible early G1 arrest, I formally test whether cell cycle progression drives loss of self-renewal capacity in culture. Long-term in vivo transplantation approaches conclusively establish that cell cycle progression is not responsible for the loss of long-term repopulation capacity associated with clinically relevant HSC culture.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Johnson, Carys

Advisor dc:contributor.advisor

• Laurenti, Elisa

• haematopoietic stem cell
• gene therapy
• haematopoiesis
• HSC
• cell cycle

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.86475

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/339064