EPCR expression within the BM SLAM LSK fraction is a high-confidence predictor of transplantation-associated HSC activity.

(A) Expression patterns of EPCR and CD41 within the BM LSK SLAM fraction. Gates depict the assessed cell fractions.

(B) Strategy used to assess the correlation of EPCR and CD41 expression to the in vivo HSC activity.

(C) Test-cell derived chimerism in PB 4 and 16 weeks post-transplantation.

(D) Test-cell derived chimerism in BM EPCR+ SLAM LSKs 16 weeks post-transplantation.

(E) Correlation between duration of radioprotection and EPCR expression levels. BM LSK SLAM cells were co-stained with EPCR and index-sorted at one cell per well, cultured for 21 days, and the content of each well transplanted to individual recipients (n = 22). Correlation to mortality of individual mice was made by assessing which well was transplanted into which mouse and coupling this to the index-sorting information. The grey dash line indicated the separation of EPCR higher (>4900) or lower expression (<4900) cHSCs.

(F) Donor contribution in PB myeloid cells. Mice were transplanted with ex vivo expanded cells from either 50 SLAM LSK EPCRlow (n = 5) or 50 SLAM LSK EPCRhigh cells (n = 5) and assessments made 16 weeks after transplantation.

All data points depict values in individual recipients. Error bars denote SEM. The asterisks indicate significant differences. *, p < 0.05; **, p < 0.01. In E) a regression line was generated based on an end-point survival of 150 days (the time at which the experiment was terminated).

The phenotypic properties of HSC expansion cultures.

(A) Frequency and fold change of phenotypic cHSCs (EPCRhigh SLAM LSKs) in ex vivo cultures after 14 or 21 days of culture. Data points depict values from individual cultures initiated from 50 cHSCs. Error bars denote SEM. The asterisks indicate significant differences. ****, p < 0.0001.

(B) UMAP (based on SAILERX dimensionality reduction) of single-cell multiome profiling of cells expanded ex vivo for 21 days. Cell type annotations were derived using marker gene signatures and distal motif identities.

(C) Trajectory analysis of lineage differentiation for cells expanded ex vivo (left), with the top 3 scoring TF motifs of each cluster (right).

(D) Expression of HSC signature on whole culture.

(E) Expression of HSC signature of EPCR+ cells sorted from ex vivo cultures.

(F) Cell cycle phase classification of EPCR+ cells sorted from ex vivo cultures.

HSC activity can be prospectively isolated from cHSC cultures and associates to a minor EPCR+ SLAM LSK fraction.

(A) Strategy to assess the in vivo HSC activity of subfractions from ex vivo cultures.

(B) Test-cell derived chimerism in PB myeloid lineages over 24-weeks post-transplantation. Data represent mean values ± SEM (n = 5 for each group). A one-way ANOVA test was applied and the asterisks indicate significant differences among the four groups. ****, p < 0.0001.

(C) Test-cell derived chimerism in BM progenitor subsets 24-weeks post-transplantation. Numbers indicate fold differences between the EPCR+ CD48-/low and EPCR+CD48+ fractions, and data points depict chimerism levels in individual recipients.

Quantification of repopulating activity from expanded cHSCs.

(A) Competitive transplantation strategies used to assess the repopulation of ex vivo expanded cHSCs.

(B) Test-cell derived PB reconstitution 16-weeks post-transplantation. Symbols denote individual mice and means ± SEM.

(C) BM cHSCs chimerism 16 weeks post transplantation. Symbols denote individual mice, and means ± SEM.

(D) Barcode approaches used to assess the clonal HSC contribution of ex vivo expanded HSCs before (i) or after (ii) ex vivo expansion.

(E) Clone sizes of unique barcodes in ‘parental’ recipients and their appearance in ‘daughter’ recipients, demonstrating extensive variation in expansion capacity among individual HSCs. Red lines indicate the median clone size in ‘parental’ recipients.

(F) Clone sizes of unique barcodes detected in BM myeloid cells of ‘parental’ recipients and their corresponding contribution in ‘daughter’ recipients. The red line denotes the correlation/linear regression.

(G) Clone sizes in ‘parental’ recipients transplanted with ‘pre-culture’ barcoded cells, or in recipients of ‘post-culture’ barcoded cells. Median clone sizes are shown with interquartile ranges.

(H) Frequency of barcodes and their clone sizes in recipients of pre- or post-cultured barcoded HSCs. BCs, barcodes.

All data points depict values in individual recipients or barcodes. Asterisks indicate significant differences. *, p < 0.05; ****, p < 0.0001.

RUs for each lineage in PB and for BM cHSCs of each recipient.

Cultured cHSCs allow for transplantation into non-conditioned syngeneic recipients.

(A) Strategy to assess the ability of ex vivo expanded cHSCs to engraft unconditioned recipients.

(B) Test-cell derived PB reconstitution 24-weeks post-transplantation. Symbols denote individual mice and means ± SEM.

(C) Strategy used to assess the in vivo proliferation of ex vivo expanded cHSCs.

(D) BM cHSC chimerism 2-8 weeks post transplantation.

(E) Representative CTV label profiles of donor EPCR+ cHSCs compared to negative control signal (host EPCR+ cHSCs) and positive signal (donor CD4+ spleen cells) at 2 or 8 weeks post-transplantation.

(F) Donor EPCR+ cHSCs were evaluated for the number of cell divisions they had undergone through 8 weeks post-transplantation.

All data points depict values in individual recipients.