EMBO/soda-nel huggingface dataset:
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 1

Figure 1. CRMP2 deficiency leads to axonal growth defects in peripheral nerves.

(A) Generation of crmp2 knockout mice by TALEN mutagenesis. Alternative first exons are highlighted in black and red.

(B) CRMP2 expression in adult cortex (layers VI - IV), corpus callosum (CC) and hippocampal area CA1. CRMP2 (green) is expressed throughout the cortex and hippocampus, CRMP2A (red) is particularly strongly expressed in a subset of callosal axons. Nuclei are counterstained with Hoechst 33342. Scale bars: 50 μm.

(C) Whole-mount immunohistochemistry with anti-neurofilaments antibody for visualization of peripheral nerves. In crmp2-/- embryos, the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated (first and second row, arrowheads). Similarly, we detected increased growth and branching of lateral branches of spinal nerves in crmp2-/- embryos (third row, arrowheads). Trigeminal nerve (V) and its branches (Op - ophthalmic, Mx - maxillar, Md - mandibular) are indicated. Scale bars: 500 μm.

(D, E) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve, normalized to WT littermates (each dot depicts one embryo). Area of ophthalmic branch in crmp2-/- was increased by 66% (p<0.05, n=10, 3 litters). For spinal nerves, total area in knockouts was increased by 45% (p<0.05, WT n=8, crmp2-/- n=7, 3 litters). Mean ± SD, * p<0.05, t-test.

(F) Growth of motor neurons from E11.5 - E12.5 WT and crmp2-/- spinal cord explants in microfluidic chambers. Excerpts from 14 hour time-lapse imaging are shown. Upon application of Sema3A into distal compartment, WT axons tend to stop or slow down their growth unlike crmp2-/- axons. Purple lines highlight the growth path of individual axons in a distal chamber. Scale bars: 100 μm. See also movie EV1.

(G) Quantification of the fraction of growing (>50 μm) axons in one imaging field (WT: control 76.8±5.7%, Sema3A 57.7±10.6%, p<0.001, crmp2-/-: control 75.4±10.4%, Sema3A 75.4±6.7%, p=0.99), n=3 experiments per genotype, 7-8 explants per condition. Mean ± SD. *** p<0.001, 2-way ANOVA with Bonferroni's multiple comparisons test.
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 2

Figure 2. CRMP2 regulates postnatal development of corpus callosum.

(A) CRMP2 deficiency leads to callosal hypoplasia. Shortening of corpus callosum (arrowheads) is apparent in both sagittal (first row) and coronal sections (second row) of adult brains. Scale bars: 1 mm.

(B) Quantification of callosal length in 30-days old mice (P30, n=3, crmp2-/- is 80.8±5% of WT, p=0.003) and adult mice (n=3, crmp2-/- is 80.5±3% of WT, p=0.002), mean ± SD, * p<0.05, t-test.

(C) Labeling of adult corpus callosum with anti-neurofilaments antibody in sagittal sections. Outline depicts missing posterior part of the tract in crmp2-/- mice. Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure (hbc), while in crmp2-/- mice callosum terminates rostrally before reaching the hbc (arrows). PC indicates posterior commissure. Scale bars: 500 µm.

(D) The growth of GFP-labeled callosal axons in the contralateral cortex at P6 (embryos were electroporated at E15.5). Note the disorganized paths of crmp2-/- axons. Scale bars: 200 μm.

(E) Schematic drawing of the callosal axon path and axonal tortuosity calculation. Pyramidal neurons in layer II/III projects their axons into the contralateral cortex (rectangle depicts the area displayed in (D)). Axonal tortuosity quantification as shown in (F), Tortuosity=1 if a=b.

(F) Quantification of tortuosity of axons (WT n=6 pups, crmp2-/- n=3 pups) upon their exit from the callosal tract (WT 1.016±0.003 vs. crmp2-/- 1.044±0.012, p<0.001), mean ± SD, ***p<0.001, t-test.

(G) DiI-labeled callosal axons from P9 oblique brain sections (see the Appendix Fig. S1) and their reconstruction in Neurolucida 360 (WT n=6, crmp2-/- n=9). Scale bars: 200 μm.

(H) Polar histograms of callosal axons reconstructed in (G). Note the broader range of axon growth angles in crmp2-/- mice.

(I) Left: schematic representation of polar histogram analysis by clustering the traced axons (WT n=6 pups, crmp2-/- n=9 pups) into 3 groups based on the growth angles. Right: proportion of axons growing in selected clusters (WT, 0°-±20°: 67.3±10.3%; ±20°-±40°: 21.2±5.3%; ±40°-±60°: 6.22±3.2%; crmp2-/-, 58.5±5%, p=0.047, 27.7±4.3%, p=0.022, 8.64±2.25%, p=0.11), mean ± SD, *p<0.05, t-test.
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 3

Figure 3. Infrapyramidal bundle fails to prune in crmp2-/- mice.

(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.

(C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm.

(E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm.

(G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 4

Figure 4. Defective pruning of corticospinal visual axons in crmp2-/- mice.

(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.

(B) Schematic drawing of DiI injection and axon tracing. Axons that initially enter pyramidal tract fail to prune in crmp2-/- mice (red line).

(C) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 5

Figure 5. CRMP2 mediates Sema3F signaling in primary neurons.

(A) Time-lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation. Upper panel: Axon growth without Semaphorin stimulation. Middle panel: stimulation with Sema3A (0 hours) (1nM, n=669 axons for WT, 761 axons for knockout) causes retraction of both WT and crmp2-/- neurons. Lower panel: stimulation with Sema3F (5 nM, n=602 axons for WT, 955 axons for knockout) causes axon retraction in WT, but not in crmp2-/- neurons. Red triangles depict growing axons, yellow retracting axons and blue asterisks indicate steady non-growing axons. See also movie EV2 and EV3. Scale bars: 100 μm (whole image field) and 50 μm (magnified).

(B) Schematic drawing of the experimental setup.

(C) Quantification of retracting axons (number of retracting vs. steady axons, 3 experiments). WT: control 13.4±5%, Sema3A 31.2±6% (p<0.001), Sema3F 36.9±8.7% (p<0.001); crmp2-/-: control 19.8±1.8%, Sema3A 28.5±4.4% (p<0.05), Sema3F 22.4±4.1% (p>0.99), mean ± SD are shown. ***p<0.001, *p<0.05, 2-way ANOVA with Bonferroni's multiple comparisons test.
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 6

Figure 6. CRMP2 regulates dendritic spine refinement in dentate gyrus granule cells.

(A, B) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm.

(C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites.

(D, E) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2-/- mice (n=3 animals, ≥25 dendrites). Quantification of granule cell branching by Sholl analysis showed no significant differences (p>0.99). Scale bars: 50 μm, mean ± SEM, 2-way ANOVA with Bonferroni correction.

(F) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies (n=5 mice/genotype). GL indicates granule cell layer, IML/OML indicate inner/outer molecular layer, respectively. Density of colocalized PSD95/VGLuT2 puncta was counted. IML: WT 109±21, crmp2-/- 156±15 (p=0.004). OML: WT 281±38, crmp2-/- 297±80 (p=0.7). Scale bars: 10 μm, mean ± SD, *p<0.05, t-test.

(G) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm.

(H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.
CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine
remodeling (2019)
Figure 7

Figure 7. Behavioral analysis of crmp2-/- mice.

(A - B) Ultrasonic vocalization was measured at P6, P8 and P12 (WT n=14 pups, crmp2-/- n=13 pups). In crmp2-/- mice, there is a significant decrease in the rate and duration of calls at P8 (call rate, WT 352±58/5 min, crmp2-/- 164±41/5 min, p=0.015; call duration, WT 9.52±2 ms, crmp2-/- 3.36±1 ms, p=0.011) and P12 (call rate, WT 46±16/5 min crmp2-/- 1.54±1.24/5 min, p=0.002; call duration, WT 0.68±0.35 ms, crmp2-/- 0.007±0.006 ms, p=0.002). Mean ± SEM, *p<0.05, t-test (P6, P8), Mann-Whitney test (P12).

(C) Representative sonograms of the P8 mice.

(D) 3-chamber test (WT n=11 mice, crmp2-/- n=13 mice). In sociability phase, WT mice spent significantly more time with a social partner (stranger 1), unlike knockouts. (WT - stranger 1: 0.14±0.01, object: 0.07±0.007 (p=0.0001); crmp2-/- - stranger 1: 0.07±0.01, object: 0.04±0.006 (p=0.07)). In social novelty phase, when an object was substituted with a second social partner (stranger 2), both WTs and knockouts preferred novel mice to known mice. (WT - stranger 1: 0.03±0.006, stranger 2: 0.09±0.008 (p<0.0001); crmp2-/- - stranger 1: 0.02±0.004, stranger 2: 0.05±0.01 (p=0.02)). Mean ± SEM, *p<0.05, ***p<0.001, t-test.

(E - G) Elevated plus maze test (n=10 mice/genotype). (E) Total distance walked is similar in WT and crmp2-/- (WT 7.9±0.45 m, crmp2-/- 8.6±0.5 m, p=0.3). (F) Frequency and (G) duration of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. (CA frequency: WT 13.7±0.9/5 min, crmp2-/- 10.5±1.1/5 min, p=0.04; OA frequency: WT 7.7±0.8/5 min, crmp2-/- 14.6±1.7/5 min, p=0.002; MID frequency: WT 20.6±1/5 min, crmp2-/- 24.6±1.6/5 min, p=0.06; Total arm visits: WT 21.4±1/5 min, crmp2-/- 25.1±1.5/5 min, p=0.07; CA duration: WT 177±17 s., crmp2-/- 75.3±12.6 s, p<0.001; OA duration: WT 42±12.6 s, crmp2-/- 144.4±21 s, p<0.001; MID duration: WT 80.26±11.5 s., crmp2-/- 80.17±12.6 s., p=0.99), mean ± SEM, *p<0.05, ***p<0.001, t-test.

(H, I) Y maze test (WT n=9 mice, crmp2-/- n=8 mice). Decreased alternations between arms of the maze indicates impaired working memory (number of visits, WT: 23.1±1.9, crmp2-/- 30.4±3.8, p=0.12; alternations, WT 68.8±2.5%, crmp2-/- 57.3±2.5%, p=0.006), mean ± SEM, *p<0.05, t-test.
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 1

Figure 1. HO-1 is expressed in BM endothelial cells and pericytes.

(A) Metaphysis region in the BM is rich in endomucin+ capillaries expressing HO-1. mp - metaphysis; gp - growth plate; scale bar 100 μm.

(B) The HO-1 positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm.

(C) HO-1 is expressed by smaller endomucin+CD31+ capillaries (#) as well as in bigger endomucin-/lowCD31+ arteries (*). CD31- pericytes wrapping the artery also express HO-1 (*); scale bar 20 μm.

(D) HO-1 positive capillaries in the metaphysis expressed CD31 and Sca-1. The capillaries are enveloped by HO-1 expressing pericytes. Part of the HO-1+ pericytes express Sca-1 (#) while others show no or low Sca-1 signal (*); scale bar 20 μm.

(E) Flow cytometry analysis revealed the highest expression of HO-1 in CD31+Sca-1+ ECs. CAR and PαS populations also express HO-1, while most of non-hematopoietic CD45-Ter119- are HO-1-negative in steady-state conditions.

(F) BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ.

(G, H) HO-1 expression on mRNA level quantified by (G) qPCR or (H) RNA-seq. qPCR analysis based on two independent experiments n = 10-11/group, and RNAseq analysis has n = 3-4/group, two-tailed t-test for two groups comparison, and one way ANOVA with Bonferroni post-test for multiple group comparison. *p< 0.05, ****p< 0.0001. Data are shown as mean ± SEM.

(I) ECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean ± SEM. *p< 0.05, ***p<0.001, two-tailed unpaired t-test.

(J) Middle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean ± SEM. **p<0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1-/- bone marrow section is provided in Appendix Fig S1.
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 2

Figure 2. HO-1 deficiency affects expression of hematopoietic factors in ECs and CARs.

(A) RNA-seq revealed 111 DEGs in HO-1-/- ECs.

(B, C) The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA.

(D) Selected GOBP terms enriched in GSEA analysis in HO-1-/- ECs.

(E, F) Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs.

(G) RNA-seq revealed 100 DEGs in HO-1-/- CARs.

(H, I) The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA.

(J) Selected GOBP terms enriched in GSEA analysis in HO-1-/- CARs.

(K, L) Heatmap of selected (K) skeletal biology and (L) hematopoietic factors expression in HO-1-/- and HO-1+/+ CARs. Correlation similarity metric and average linkage clustering were used in the presented heatmaps.

Data information: Color key on the heatmaps represents gene expression (as z-score among row) red - high expression, blue - low expression
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 3

Figure 3. HO-1-/- mice have disturbed frequency of ECs, CARs, and MQs in BM and altered production of hematopoietic cytokines.

(A) HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ.

(B) Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice.

(C, D) Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs.

(E) Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.

(F, G) The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.

Data information: Where individual data points not shown n = 4/group; unpaired two-tailed t-test. *p< 0.05, **p<0.01, ***p<0.001. Data shown are mean ± SEM from a single experiment.
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 4

Figure 4. HO-1 deficiency causes loss of LT-HSC quiescence and expansion of stem cell pool.

(A) Gating strategy of hematopoietic stem and progenitor cells.

(B-D) Young HO-1-/- mice possess higher number of (B) LT-HSCs, (C) ST-HSCs and (D) MPPs.

(E) Exemplary analysis of cell cycle with Ki67 and nuclear dye.

(F) More young HO-1-/- LT-HSCs are in G1 and S/G2/M cell cycle phases. The presented cell cycle analysis is from two independent experiments.

(G) Young HO-1-/- MPPs do not differ in cell cycling from young HO-1+/+ MPPs. The presented cell cycle analysis is from two independent experiments.

(H) More old HO-1-/- LT-HSCs are in G1 phase in comparison to old HO-1+/+ LT-HSCs, but not in S/G2/M phase. The presented cell cycle analysis is from two independent experiments.

(I) Old MPPs do not differ in cell cycling between genotypes. The presented cell cycle analysis is from two independent experiments.

(J) Young HO-1-/- LT-HSCs contain more γH2aXhigh cells than young HO-1+/+, 378-991 cells analyzed from 7 mice/group.

(K) Young HO-1-/- MPPs contain more γH2aXhigh cells than young HO-1+/+, 1693-2790 cells analyzed from 7 mice/group.

(L, M) Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.

(N) HSCs from young HO-1-/- mice provide worse hematopoietic reconstitution after transplantation than HO-1+/+ HSCs. Data are shown as mean ± SEM, n = 8-9 mice/group.

Data information: For comparison of two groups unpaired, two-tailed t-test was used. For contingency analysis (J and K) Fisher exact test was applied. For 2 variables comparisons (N) 2-way Anova with Bonferroni post-test was used. *p< 0.05, **p<0.01, ***p<0.001, ****p< 0.0001
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 5

Figure 5. LT-HSCs from young HO-1-/- mice possess transcriptional profile resembling aged LT-HSCs.

(A) RNA-seq revealed 1067 DEGs between young HO-1-/- and HO-1+/+ LT-HSCs.

(B) RNA-seq revealed 595 DEGs between old HO-1-/- and HO-1+/+ LT-HSCs.

(C) Selected GOBP terms enriched in GSEA analysis in young HO-1-/- LT-HSCs.

(D) Selected GOBP terms enriched in GSEA analysis in old HO-1-/- LT-HSCs.

(E) Significant part of DEGs (31% total) in young HO-1-/- LT-HSCs overlaps with DEGs identified in LT-HSCs during physiological aging.

(F) PC1 in PCA analysis on selected 1148 genes separated young HO-1+/+ LT-HSCs from other groups, clustering the young HO-1-/- LT-HSCs together with old LT-HSCs.

(G) Hierarchical clustering on the 20% of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO-1+/+ LT-HSCs in one and old LT-HSCs with young HO-1-/- LT-HSCs in second.

(H) Selected GOBP terms enriched in GSEA analysis based on 297 identified genes.

Data information: Color key on the heatmaps represents gene expression (as z-score among row).
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 6

Figure 6. HO-1 deficiency in the niche causes exhaustion of LT-HSCs.

(A) Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status.

(B) Scheme of the experiment assessing long-term effect of HO-1 deficient niche on function of HSCs.

(C) Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients. *p< 0.05, 2-way ANOVA. Data are shown as mean ± SEM, n = 5-12/group

(D, E) (D) Chimerism among BM HSPCs fractions and (E) total number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group

(F) HSCs that were initially transplanted to primary HO-1-/- recipients did not reconstitute secondary HO-1+/+ recipients, in contrast to HSCs that were initially transplanted to HO-1+/+ recipients, n = 5-12/group.

(G) Scheme of the experiment assessing short-term homing of HSPC to HO-1 deficient niche.

(H) Short term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 7

Figure 7. HO-1+/+ expression in the recipients restores function and transcriptional profile of HO-1-/- LT-HSCs.

(A) Scheme of the experiment verifying if the HO-1+/+ niche is able to reverse phenotype of HO-1-/- LT-HSCs.

(B) Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group.

(C, D) Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerism and (D) BM LT-HSC chimerism. Data are shown as mean ± SEM, n = 7-8 mice/group.

(E) Analysis pipeline used to determine whether transplantation of HO-1-/- HSCs to the HO-1+/+ recipients reverses their transcriptional alterations.

(F) Overlap between DEGs in young HO-1-/- HSCs and DEGs changed by transplantation alone. 145 overlapping genes were excluded from further analysis.

(G) GSEA analysis based on 145 excluded genes indicates processes that cannot be analyzed with the pipeline.

(H) Only 1 out of 267 DEGs identified in non-transplanted HO-1-/- LT-HSCs was still dysregulated in HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+.

(I) Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations.

(J) GSEA analysis based on genes that were altered in non-transplanted HO-1-/- LT-HSCs, but were normalized by double transplantation to the wild-type niche.
Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells (2019)
Figure 8

Figure 8. HO-1 deficiency in MSCs affects growth and differentiation of HSC-derived hematopoietic colonies.

(A) Scheme of experiment based on HSC co-culture with MSCs and colony formation from single cells in differentiation media.

(B) Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test.

(C-F) PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+ and (F) cells lacking expression of analyzed lineage markers (Lin-).

(G,H) Analysis of frequency of colonies derived from HO-1+/+ or from HO-1-/- MSC co-cultures among identified clusters. Enrichment in cluster analyzed by Fisher exact test, **p<0.01, two-tailed unpaired t-test.

(I) Sdf1α mRNA levels in BM of HO-1+/+ or HO-1-/- mice after administration of Noggin. Data are shown as mean ± SEM. *p< 0.05, two-tailed unpaired t-test, n = 10/group

(J, K) (J) LT-HSC frequency and (K) cell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice. Data are shown as mean ± SEM. *p< 0.05, **p<0.01, two-tailed unpaired t-test, n = 8-10/group

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