Fig 2: SPINK2 is highly expressed in CD34+ bone marrow cells(A) Uniform manifold approximation and projection (UMAP) graph shows the CD34+ cell clusters within human bone marrow as annotated by Setty et al.19(B) UMAP graph showing SPINK2 expression in CD34+ cells.(C) SPINK2 and SPINK9 expression values are reported as averaged normalized CPM (top graph), and percentage of SPINK2 or SPINK9 positive cells in different population of CD34+ cells within the bone marrow (bottom graph). A cell is considered positive if normalized CPM value is > 0. Numbers of analyzed cells (n) in each population are the following: HSC, hematopoietic stem cell (n = 4690); HMP, hematopoietic multipotent progenitor (n = 4306); CMP, common myeloid progenitor (n = 2328); GMP, granulocyte-monocyte progenitor (n = 3713); DP, dendritic progenitor (n = 2075); MP, megakaryocyte progenitor (n = 507); EP, erythroid progenitor (n = 3463); CLP, common lymphoid progenitor (n = 3237); (D) Averaged normalized CPM (left graph) and percentage of SPINK positive cells (right graph) in different populations of hematopoietic cells within the bone marrow of mouse C57BL/6. LT-HSC, Long Term-HSC (n = 216); HSCs/HMPs, hematopoietic stem and progenitor cells (n = 852); MP/EP/CMP/GMP, megakaryocyte progenitor/erythroid progenitor/common myeloid progenitor/granulocyte-monocyte progenitor (n = 851).

Fig 3: The temporary inhibition-diffusion model for SPINK2 in hematopoietic stem cell niche(A) The concentration of the complex tPRSS SPINK2, indicated as [EI] and shown in top graphs, and the velocity of conversion of protein substrate (Sv), shown in bottom graphs, were calculated at different distances from the source cell using different values of D (µ2/sec), as indicated above each plot. Results obtained for temporary or persistent inhibitor are shown in each plot. A SPINK2/tPRSS ratio = 2 is used in these simulations.(B) The velocity of conversion of protein substrate (Sv) per unit volume was calculated at different distances from the source cell using different ratios [I]/[E] at the source boundary, as indicated above each plot. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A D value = 5 µ2/sec is used in these simulations.(C) The concentration of the complex [EI] is shown in top panels, and the velocity of conversion of protein substrate (Sv) per unit volume in bottom panels. A fixed total concentration of [E] is used in left panels, and an inverse concentration gradient in the right panels. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A SPINK2/tPRSS ratio = 2 at the boundary of inhibitor source and a D value = 5 µ2/sec are used in these simulations.

Fig 4: SPINK2 transcript and protein are highly expressed in CD34+ bone marrow cells(A) qRT-PCR by 2−ΔΔCT method confirms high levels of SPINK2 in purified CD34+ cells. ELANE, typically expressed in differentiated neutrophil granulocytes and granulocyte-monocyte progenitors, has been evaluated as negative control.(B) SPINK2 and PRSS57 are highly expressed at protein level in CD34+ HSPCs. Protein intensity of SPINK2 and PRSS57 is expressed as normalized label-free (LF) score in CD34+ HSPC (Hennrich et al.22). Negative control results in bone marrow mesenchymal stromal cells (MSC) are also shown.(C) Western blot showing SPINK2 enrichment in CD34+ cells. GAPDH and ACTB were the loading control.(D) SPINK2 (P20155) and SPINK1 (P00995) structures have been downloaded from AlphaFold Protein Structure Database developed by DeepMind and EMBL-EBI (https://alphafold.ebi.ac.uk/entry/P20155; https://alphafold.ebi.ac.uk/entry/P00995).23,24 Kazal domain is highlighted in green square.(E) SPINK2 (NP_066937, 84 amino acids) and SPINK1 (NP_001366539) protein alignment was performed by Constrain-based Multiple Alignment tool (Cobalt, https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi), and as showed in Figure 2E the method highlights in red color highly conserved aminoacidic positions based on the relative entropy threshold of the residue, while blue color indicates lower conservation. Kazal domain is underlined (green) and is located among amino acids in position 36 and 84 and includes 6 typical cysteines forming three pairs of disulfide bonds that stabilize its conformation.

Fig 5: Expression of putative tPRSS in human bone marrowAverages of normalized CPM values (top graph) and percentage of positive cells (bottom graph) in different CD34+ cell subpopulations for (A) PRSS1, PRSS2, and PRSS3; (B) PRSS27, PLAT, and DPP7; and (C) PRSS57, CTSG, ELANE, and PRTN3.(D) qRT-PCR analysis of PRSS1, PRSS2, and PRSS57 transcripts in purified mobilized CD34+ blood cells. CD34-cells were used as calibrator for fold change calculation.(E) SPINK2/PRSS57 or SPINK2/PRSS2 ratios in different subpopulations of HSPCs.(F) Percentage of PRSS2+ and PRSS57+ cells in SPINK2+ cell population.(G) Percentage of SPINK2+ cells in PRSS2+ cell population and in PRSS57+ cell population.