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, 25 (5), 776-783

Highly Efficient Therapeutic Gene Editing of Human Hematopoietic Stem Cells

Highly Efficient Therapeutic Gene Editing of Human Hematopoietic Stem Cells

Yuxuan Wu et al. Nat Med.

Abstract

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

Conflict of interest statement

Competing Interests Statement

The authors declare competing financial interests, Y.W., J.Z., S.A.W., D.E.B. have applied for patents related to therapeutic gene editing including US Patent Applications 13/72236, 15/572,523, 18/34618, 18/43073.

Figures

Extended Data Figure 1 |
Extended Data Figure 1 |. Cas9 RNP dose dependent editing of BCL11A enhancer for HbF induction in CD34+ HSPCs.
a, Comparison of indel frequencies with in vitro transcribed (IVT), synthetic (syn) and modified synthetic (MS) sgRNAs in CD34+ HSPCs by TIDE analysis. b, Comparison of viability of CD34+ HSPCs edited with in vitro transcribed (IVT), synthetic (syn) and modified synthetic (MS) sgRNAs. c, Dose dependent editing rates with Cas9 coupled with MS-sgRNA-1617 and −1639 targeting BCL11A enhancer and -e2 targeting BCL11A exon2 in CD34+ HSPCs by TIDE analysis. d, Comparison of indel frequencies with different molar ratios of Cas9 to MS-sgRNA in CD34+ HSPCs by TIDE analysis. e, Comparison of viability of CD34+ HSPCs edited with different molar ratios of Cas9 to MS-sgRNA. f. Percent HbF+ cells by flow cytometry analysis in erythroid cells in vitro differentiated from CD34+ HSPCs edited by RNP coupled with various sgRNAs (each targeting BCL11A enhancer). Error bars indicate standard deviation (n = 3 replicates). g, Summary of deep sequencing data derived from the Cas9 RNP (coupled with MS-sgRNA-1617) edited CD34+ HSPCs. Asterisk indicates unedited allele. h, HbF induction by HPLC analysis in erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs. i, Genotyping and β-like globin expression analysis of clonal erythroid cells derived from single CD34+ HSPCs. Error bars indicate standard deviation (n = 3 technical replicates per colony). j, BCL11A expression in CD34+ HSPCs edited with Cas9 coupled with various MS-sgRNAs targeting BCL11A enhancer. Expression normalized to CAT, measured by RT-qPCR on day 11 of in vitro differentiation. Error bars indicate standard deviation (n = 3 replicates). k, Correlation of γ-globin mRNA expression determined by RT-qPCR versus HbF by HPLC. Black dots represent samples edited with 2xNLS-Cas9 coupled with various MS-sgRNAs. l, Correlation of BCL11A mRNA versus γ-globin mRNA determined by RT-qPCR. Black dots represent samples edited with 2xNLS-Cas9 coupled with various sgRNAs. m, n, Genotyping and HbF level by HPLC of clonal erythroid cells derived from single CD34+ cells from two independent healthy donors (βAβA#1 in m and βAβA#3 in n) edited with MS-sgRNA-1617. o, Correlation of percent γ-globin mRNA determined by RT-qPCR versus HbF by HPLC. Black dots represent single colonies edited with 2xNLS-Cas9 coupled with MS-sgRNA-1617. The Pearson correlation coefficient (r) is shown. In all panels, data are plotted as mean ± SD. Data are representative of three biologically independent replicates.
Extended Data Figure 2 |
Extended Data Figure 2 |. Indel frequencies from deep sequencing.
a. Frequency distribution of alleles with and without indels (shown in blue and red respectively) from deep sequencing of CD34+ HSPCs edited with 2xNLS-Cas9 RNP with indicated MS-sgRNAs targeting BCL11A enhancer. b, Correlation of indel frequencies by deep sequencing versus indel frequencies by TIDE analysis. The Pearson correlation coefficient (r) is shown.
Extended Data Figure 3 |
Extended Data Figure 3 |. Long-term multi-lineage engraftment of BCL11A enhancer edited HSPCs in immunodeficient mice.
CD34+ HSPCs from two healthy donors were electroporated with 2xNLS-SpCas9 RNP (coupled with MS-sgRNA-1617) and transplanted into NBSGW mice. Non-electroporated cells were transplanted as controls. 0.4 million cells per mouse were infused for donor βAβA#1, and 0.8 million cells per mouse for donor βAβA#2. a, Mouse bone marrow (BM) was analyzed for human cell chimerism by flow cytometry 16 weeks after transplantation, defined as %hCD45+/(%hCD45++%mCD45+) cells. Each symbol represents a mouse, and mean for each group is shown. b, Indels at the human BCL11A enhancer were determined by TIDE analysis in the input HSPCs prior to transplant and in the mouse bone marrow 16 weeks after transplant. Each engrafted dot represents one mouse, and mean for each group is shown. c, BM collected 16 weeks after transplantation was analyzed by flow cytometry for multilineage reconstitution (calculated as percentage of hCD45+ cells). d, BM collected 16 weeks after transplantation was analyzed by flow cytometry for CD235a+ erythroid cells (calculated as percentage of mCD45hCD45 cells). e-g, Gene expression analysis by RT-qPCR in human cells (from donor βAβA#2) from BM of engrafted mice. BCL11A expression normalized by CAT in human B cells (e) or human erythroid cells (f) sorted from BM of engrafted mice, and β-like globin expression (g) by RT-qPCR in human erythroid cells sorted from BM. h, BM from one engrafted mouse with unedited control or edited cells (from donor βAβA#1) were transplanted to three secondary NBSGW mice each (control mouse shown with black circle and edited mouse with green diamond symbol in (a, b, d). After 16 weeks, BM was analyzed for human cell chimerism by flow cytometry. i, Indel frequencies within human BCL11A enhancer in BM 16 weeks after secondary transplantation. Each symbol represents an individual recipient mouse. Data are plotted as mean ± SD for (c). Median of each group with 2 to 4 mice is shown as line for the other panels.
Extended Data Figure 4 |
Extended Data Figure 4 |. Highly efficient editing of BCL11A enhancer in CD34+ HSPCs.
a, Dose dependent viability enhancement with glycerol or glycine after electroporation. 0.27 M = 2% glycerol, 0.2 M = 1.5% glycine. b, Quantification of editing frequency from deep sequencing of CD34+ HSPCs edited with 3xNLS-Cas9 RNP with MS-sgRNA-1617. c, Length distribution of alleles with and without indels (shown in blue and red respectively) from deep sequencing of the 2xNLS-Cas9 RNP with ms-sgRNA-1617. d, e, Reduction of BCL11A mRNA by RT-qPCR or protein by western blot after editing of human BCL11A enhancer with 2xNLS-Cas9 or 3xNLS-Cas9 RNP with MS-sgRNA-AAVS1 or −1617 on various days of in vitro differentiation. Relative areas under curve (AUCs) are indicated. f, g, β-like globin expression by RT-qPCR and HbF level by HPLC in erythroid cells in vitro differentiated from 3xNLS-Cas9 RNP coupled with MS-sgRNA-1617 edited CD34+ HSPCs. All data represent the mean ±SD. Statistically significant differences are indicated as follows: *P < 0.05 as determined by unpaired t test. P= 0.0152 for (f) and 0.0443 for (g). In all panels, data are plotted as mean ± SD and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.
Extended Data Figure 5 |
Extended Data Figure 5 |. Long-term multi-lineage reconstituting HSCs edited with 3xNLS-Cas9.
a-d, NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited healthy donor CD34+ HSPCs 2 h (day 0), 24 h (day 1) or 48 h (day 2) after electroporation. BM were collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (a), multilineage reconstitution (b) or human erythroid cells (c) in BM, as well as indel frequencies determined by TIDE analysis (d). e-h, NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited healthy donor CD34+ HSPCs supplemented with 2%, 4% or 6% of glycerol for electroporation. BM were collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (e), multilineage reconstitution (f) or human erythroid cells (g) in BM, as well as the indel frequencies determined by TIDE analysis (h). i, Multilineage reconstitution analysis of BM collected from mice engrafted with control or edited CD34+ HSPCs (from donor βAβA#4). Error bars indicate standard deviation. Data are plotted as mean ± SD for (b, f, i). Median of each group with 1 to 3 mice is shown as line for the other panels.
Extended Data Figure 6 |
Extended Data Figure 6 |. Off-target analysis of human CD34+ HSPCs edited by SpCas9 RNP targeting BCL11A enhancer.
a, Off-target sites detected by CIRCLE-seq for MS-sgRNA-1617 targeting human BCL11A enhancer. b, Deep sequencing analysis of potential off-target sites detected by CIRCLE-seq or in silico computational prediction within human CD34+ HSPCs edited by 2xNLS-Cas9 or 3xNLS-Cas9 RNP (coupled with MS-sgRNA-1617) targeting BCL11A enhancer. On-target sequence is at the BCL11A enhancer. Dotted line at 0.1% denotes sensitivity of deep sequencing to detect indels. c, RT-qPCR analysis of p21 expression after gene editing. Relative expression to GAPDH is shown. Data are plotted as mean ± SD and representative of three biologically independent replicates.
Extended Data Figure 7 |
Extended Data Figure 7 |. Editing of BCL11A enhancer in SCD patient (βSβS) HSPCs.
NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited βSβS#1 CD34+ HSPCs 24 h (day 1) or 48 h (day 2) after electroporation. BM were collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (a), multilineage reconstitution (b) or human erythroid cells (c) in BM, as well as the indel frequencies determined by TIDE analysis (d). Error bars indicate standard deviation. e, Editing efficiency of 3xNLS-Cas9 coupled with MS-sgRNA-AAVS1 for control and −1617 for BCL11A enhancer editing in βSβS#2 CD34+ HSPCs as measured by TIDE analysis. f, β-like globin expression by RT-qPCR analysis in erythroid cells in vitro differentiated from RNP edited βSβS#2 CD34+ HSPCs. Error bars indicate standard deviation (n = 3 replicates). g, Multilineage reconstitution analysis of BM collected from mice engrafted with control or edited CD34+ HSPCs (from donor βSβS#2). h, Analysis of in vitro sickling of unedited control or edited enucleated βSβS#2 erythroid cells. Images were taken every 1 minute after MBS treatment. Result shown as percent sickled cells at each time point. Data are plotted as mean ± SD for (b, e, f, g). Median of each group with 1 to 3 mice is shown as line for the other panels.
Extended Data Figure 8 |
Extended Data Figure 8 |. Summary of engraftment analysis.
a, Indel frequencies of indicated input HSPCs and engrafted human cells in 16 week BM. b. Correlation between input cell number and human engraftment rates in 16 week BM. c, Correlation of BCL11A mRNA versus γ-globin mRNA determined by RT-qPCR. Black dots represent erythroid cells from CD34+ HSPCs edited with SpCas9 coupled with various sgRNAs differentiated in vitro without engraftment; red dots represent erythroid cells sorted from mice BM engrafted from human CD34+ HSPCs edited with SpCas9 coupled with MS-sgRNA-1617. The Pearson correlation coefficient (r) is shown. d, BM cells (engrafted from donor βAβA#1 and βSβS#1) collected from engrafted mice were in vitro differentiated to human erythroid cells for HbF level analysis by HPLC. Each dot represents erythroid cells differentiated from BM of one mouse, and mean ± SD for each group is shown. e, Relative loss of indels in HSC-enriched CD34+ CD38- CD90+ CD45RA- sorted population as compared to bulk pre-sorted HSPCs after editing by 2 μM or 5 μM RNP. All data represent the mean ±SD. Statistically significant differences are indicated as follows: ****P < 0.0001, **P < 0.01 (P=0.0046) as determined by unpaired t test. f. Comparison of β-like globin expression by RT-qPCR between erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs (pre-engraftment) and engrafted bone marrow (post-engraftment). Statistically significant differences are indicated as follows: ****P < 0.0001, ***P < 0.001 (P=0.0006), **P < 0.01 (P=0.0092) as determined by unpaired t test. In all panels, data are plotted as mean ± SD and analyzed using unpaired two-tailed Student’s t tests. Data are from indicated number of mice for (a, b, d) or representative of three biologically independent replicates for (c, e, f).
Extended Data Figure 9 |
Extended Data Figure 9 |. Indel spectrums of engrafted bone marrow and corresponding input cells.
Indel spectrums of engrafted bone marrow (BM) and corresponding input cells from four donors electroporated with 2xNLS-Cas9 or 3xNLS-Cas9 coupled with MS-sgRNA-1617 (a) or -AAVS1 (b) supplemented with different concentration of glycerol (0%G to 6%G). c, Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ in mice BM 16 weeks after transplant. The indel spectrum was determined by TIDE analysis. Indel length from −8 to +6 bp was calculated as NHEJ, and from −9 to −20 bp as MMEJ. These data comprise 28 mice transplanted with 8 BCL11A enhancer edited inputs and 5 mice transplanted with 2 AAVS1 edited inputs. Median of each group is shown as line, **P < 0.005, ****P < 0.0001 as determined by Kolmogorov–Smirnov test. d, e, Summary of most frequent indels by deep sequencing of bone marrow cells from primary recipient (d) and secondary recipient (e) engrafted with BCL11A enhancer edited CD34+ HSPCs. Asterisk indicates unedited allele. f, g, Indel spectra of HSPCs stained and sorted 2h after RNP electroporation with 3xNLS-Cas9 with sgRNA-AAVS1. HSPCs prestimulated for 24h prior to electroporation. HSPCs stained with CD34, CD38, CD90, CD45RA in (f) and with Pyronin Y, Hoechst 33342 in (g). Indels determined by Sanger sequencing with TIDE analysis after culturing cells for 4 days after sort. Data are representative of three biologically independent replicates.
Extended Data Figure 10 |
Extended Data Figure 10 |. Flow cytometry of CD34+ HSPCs with 24 hours of culture.
Cryopreserved G-CSF mobilized CD34+ HSPCs were stained with CD34, CD38, CD90, and CD45RA antibodies (in a), or stained with Hoechst 33342 and Pyronin Y (in b) at 0 hours following thaw or after 24 hours in culture with SCF, TPO and FLT3-L. HSPCs were electroporated with RNP with 3x-NLS-SpCas9 with BCL11A enhancer or AAVS1 targeting sgRNA. After 2 hour recovery, cells were stained with CD34, CD38, CD90, and CD45RA or with Hoechst 33342 and Pyronin Y, and sorted according to gates as shown in c-d.
Figure 1 |
Figure 1 |. Identification of efficient BCL11A enhancer guide RNAs for HbF induction and amelioration of β-thalassemia.
a, Eight modified synthetic (MS) sgRNAs targeting BCL11A enhancer DHS h+58 functional core marked with blue arrows. GATA and Half E-box motifs marked respectively with red or green. b, Editing efficiency of Cas9 coupled with various sgRNAs (each targeting BCL11A enhancer with exception of AAVS1) in CD34+ HSPCs measured by TIDE analysis. c, β-like globin expression by RT-qPCR analysis in erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs. d, Correlation of BCL11A mRNA expression determined by RT-qPCR versus HbF by HPLC. Black dots represent samples edited with Cas9 coupled with different sgRNAs. The Pearson correlation coefficient (r) is shown. e, Editing efficiency as measured by TIDE analysis of Cas9:sgRNA RNP targeting AAVS1 or BCL11A DHS h+58 functional core (Enh) with MS-sgRNA-1617 in CD34+ HSPCs from β-thalassemia patients or healthy donors (βAβA) of indicated β-globin genotypes. f-h, β-like globin expression by RT-qPCR normalized by α-globin (P = 0.00017 for BCL11A enhancer as compared to AAVS1 edited for all comparisons as determined by unpaired two-tailed Student’s t tests), and HbF induction by HPLC analysis in erythroid cells in vitro differentiated. i, Enucleation of in vitro differentiated erythroid cells. j, Cell size measured by relative forward scatter intensity. k, Representative microscopy image showing rounder and more uniform appearance of enucleated erythroid cells following BCL11A enhancer editing. Blue arrow indicates poikilocytes. Bar = 15 μm. l, m, Imaging flow cytometry was used to establish a circularity index (l) and then quantify (m) circularity of enucleated erythroid cells. Bar = 5 μm. In all panels, data are plotted as mean ± SD and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.
Figure 2 |
Figure 2 |. Highly efficient BCL11A enhancer editing in HSCs.
a, Schematic of 3xNLS-SpCas9 protein (1425 aa), with a c-Myc-like nuclear localization signal (NLS) at the N-terminus and SV40 and Nucleoplasmin NLSs at the C-terminus. b, Dose-dependent editing of human BCL11A enhancer with 2xNLS-Cas9 or 3xNLS-Cas9 RNP. c, Viability of CD34+ HSPCs after electroporation with 2xNLS-Cas9 and 3xNLS-Cas9. d, Viability of CD34+ HSPCs after electroporation with RNP and glycerol. e, Indel frequencies of CD34+ HSPCs after electroporation with RNP and glycerol. Error bars indicate standard deviation (n = 3 replicates with three independent healthy donors in b-e). f, Summary of most frequent indels by deep sequencing following 3xNLS-Cas9 RNP BCL11A enhancer editing of CD34+ HSPCs. Asterisk indicates unedited allele. g, Western blot analysis showing reduction of BCL11A protein after editing of human BCL11A enhancer with 2xNLS-Cas9 or 3xNLS-Cas9 RNP (MS-sgRNA-AAVS1 or MS-sgRNA-1617) at indicated days of in vitro differentiation. Blots are cropped, BCL11A observed at ~120 kDa, GAPDH at ~37 kDa. h-j, NBSGW mice transplanted with 3xNLS-Cas9 RNP (coupled with MS-sgRNA-1617) edited CD34+ HSPCs from three independent healthy donors (βAβA#1, βAβA#4 and βAβA#5). BM collected 16 weeks after transplantation were analyzed by flow cytometry for human cell chimerism (h), multilineage reconstitution from βAβA#1 (i) in BM, as well as the indel frequencies determined by TIDE analysis (j). k-m, RT-qPCR analysis of BCL11A expression in sorted human B cells (k) or human erythroid cells (l) and β-like globin expression in sorted human erythroid cells (m) from NBSGW mice transplanted with 3xNLS RNP edited CD34+ HSPCs. n, BM from one mouse each engrafted with unedited control or edited cells (βAβA#1) were transplanted to secondary NBSGW mice and BM was analyzed for human cell chimerism after 16 weeks. o, Indel frequencies within human BCL11A enhancer in BM 16 weeks after secondary transplantation. Median of each group with 3 to 9 mice in h, j-o is shown as line. Data are plotted as mean ± SD for (b-e, i) and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.
Figure 3 |
Figure 3 |. Editing BCL11A enhancer in SCD patient HSCs prevents sickling.
a, Editing efficiency of 3xNLS-Cas9 coupled with MS-sgRNA-AAVS1 for control and −1617 for BCL11A enhancer editing in βSβS CD34+ HSPCs as measured by TIDE analysis. b, β-like globin expression in erythroid cells in vitro differentiated. Error bars indicate standard deviation (n = 3 replicates). c, Genotyping and β-like globin expression analysis of erythroid cells derived from single colonies derived from unedited (ctr) or edited βSβS CD34+ HSPCs. Error bars indicate standard deviation (n = 3 technical replicates per colony). d, e, NBSGW mice were transplanted with 3xNLS-Cas9 RNP (coupled with MS-sgRNA-1617) edited βSβS CD34+ HSPCs from two independent donors (βSβS#1 and βSβS#2). BM were collected 16 weeks after transplantation and analyzed for human cell chimerism (d) in BM, as well as the indel frequencies determined by TIDE analysis (e). f-h, RT-qPCR analysis of BCL11A expression in sorted human B cells (f) or human erythroid cells (g) and β-like globin expression in human erythroid cells sorted from BM (h). i, BM from one mouse each engrafted with unedited control or edited cells (βSβS#1) from control mouse shown with black circle and edited mouse with blue triangle symbols in (d, e)) were transplanted to four secondary NBSGW mice. After 16 weeks, BM was analyzed for human cell chimerism by flow cytometry. j, Indel frequencies within human BCL11A enhancer in BM 16 weeks after secondary transplantation. Median of each group with 3 to 4 mice in d-j is shown as line. k, Phase-contrast microscopy imaging of enucleated erythroid cells in vitro differentiated from BM of NBSGW mice transplanted with unedited or BCL11A enhancer edited βSβS#1 CD34+ HSPCs with and without sodium metabisulfite (MBS) treatment. Cells with sickled cell morphology are indicated with red arrows. Bar = 10 μm. l, Analysis of in vitro sickling. Images were taken every 1 minute after MBS treatment. Result shown as percent sickled cells at each time point. Data are plotted as mean ± SD for (a-c) and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.
Figure 4 |
Figure 4 |. Persistence of NHEJ repaired alleles in HSCs.
a), Correlation of indel frequencies of input HSPCs to indel frequencies of engrafted human cells in mice BM after 16 weeks. Each dot represents average indel frequencies of mice transplanted with the same input HSPCs. Legend denoting transplant is same as in (c). The Pearson correlation coefficient (r) is shown. b, Indel spectrum of input cells from healthy donor βAβA#2 electroporated with 2xNLS-Cas9 (coupled with sgRNA-1617) supplemented with 2% glycerol and engrafted 16 week BM human cells. c, Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ in mice BM 16 weeks after transplant. The indel spectrum was determined by deep sequencing analysis. Indel length from −8 to +6 bp was calculated as NHEJ, and from −9 to −20 bp as MMEJ. These data comprise 28 mice transplanted with 8 BCL11A enhancer edited inputs and 5 mice transplanted with 2 AAVS1 edited inputs. Median of each group is shown as line, **P < 0.005, ****P < 0.0001 as determined by Kolmogorov–Smirnov test. d-e, Indel spectra of HSPCs stained and sorted 2h after RNP electroporation with 3xNLS-Cas9 with sgRNA-1617. HSPCs prestimulated for 24h prior to electroporation. HSPCs stained with CD34, CD38, CD90, CD45RA in (d) and with Pyronin Y, Hoechst 33342 in (e). Indels determined by Sanger sequencing with TIDE analysis after culturing cells for 4 days after sort. Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ at BCL11A enhancer and AAVS1 in sorted enriched HSCs (f) or G0 phase cells (g) shown. Data are plotted as mean ± SD for (f, g) and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

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