Identification of a New Cell Population Constitutively Circulating in Healthy Conditions and Endowed with a Homing Ability Toward Injured Sites

Abstract

Stem and progenitor cells are the critical units for tissue maintenance, regeneration, and repair. The activation of regenerative events in response to tissue injury has been correlated with mobilization of tissue-resident progenitor cells, which is functional to the wound healing process. However, until now there has been no evidence for the presence of cells with a healing capacity circulating in healthy conditions. We identified a rare cell population present in the peripheral blood of healthy mice that actively participates in tissue repair. These Circulating cells, with a Homing ability and involved in the Healing process (CH cells), were identified by an innovative flowcytometry strategy as small cells not expressing CD45 and lineage markers. Their transcriptome profile revealed that CH cells are unique and present a high expression of key pluripotency- and epiblast-associated genes. More importantly, CH-labeled cells derived from healthy Red Fluorescent Protein (RFP)-transgenic mice and systemically injected into syngeneic fractured wild-type mice migrated and engrafted in wounded tissues, ultimately differentiating into tissue-specific cells. Accordingly, the number of CH cells in the peripheral blood rapidly decreased following femoral fracture. These findings uncover the existence of constitutively circulating cells that may represent novel, accessible, and versatile effectors of therapeutic tissue regeneration.

Figure 1. Small-sized Lin^negCD45^negVybr^pos cells circulate in healthy mice and their percentage in the peripheral blood decreases after fracture induction.

(a–f) Representative flow cytometry strategy used to identify Lin^negCD45^negVybr^posSytox^neg (CH) cells in the peripheral blood of C57Bl/6 mice. DG, dimensional gate; LEU, leukocytes (internal positive control). (g) DAPI staining (left panel) and overlay of DAPI and bright-field (right panel) on post-sorted CH cells. Magnification 60X; scale bar, 20 μm. (h) Histogram shows the percentage of CH cells identified in the peripheral blood of 20 naive mice (n = 5 independent experiments) and 60 fractured mice after 16, 24, and 72 hours post-damage induction (20 mice/considered time point; n = 5 independent experiments). Data are presented as mean ± SD. *P = 0.0105; **P = 0.013; ***P = 0.0004.

Figure 2. Characterization of CH cells by genome-wide microarrays.

(a) Dendrograms show the hierarchical clustering, based on differentially expressed genes, of embryonic stem cell (ESC) line, ESC primary culture, c-kit⁺Sca-1⁺lineage marker⁻ cells (KSL), CD34^neg KSL, CD34^pos KSL, Lineage^neg cells, hemangioblasts (HEM), very small embryonic-like stem cells (VSEL), multipotent adult progenitor cells (MAPC), mesenchymal stem cells (MSC), and CH cells. (b) Principal component analysis of the 5000 genes with highest standard deviation for the samples mentioned in panel (a). (c) Volcano plot shows gene expression differences between CH cells and MSC. Log 2 gene ratios are plotted against negative log 10 P values.

Figure 3. CH cells express key pluripotency genes and, characteristically, genes expressed at the epiblast stage during embryo development.

(a–d) Analysis of the normalized array intensity levels of the stemness genes Sox2 (a), Oct4 (b), Nanog (c), and Klf4 (d) in the above mentioned cell populations. (e-h) The significant expression of the stemness genes in CH cells was confirmed by quantitative PCR analysis, comparing the gene expressions with MSC and ESC. (e) *P = 0.0142; ***P < 0.0007; ****P < 0.0001. (f) *P = 0.0225; **P = 0.0016; ****P < 0.0001. (g) ***P < 0.0003; ****P < 0.0001. (h) ***P < 0.0004. (i) Heatmap shows the differential expression of the main pluripotency-associated genes between ESC, MSC, and CH cells. (j) Heatmap shows the comparison of developmental-specific gene expressions among HEM, MSC, MAPC, ESC, VSEL, and CH. In the case of genes that were represented in the arrays by multiple probe sets, the signal intensity of all present probes is displayed. EMT, epithelial-mesenchymal transition; PS, primitive streak.

Figure 4. RFP^pos CH cell engraftment within the hard callus.

(a) Schematic depicting systemic cell transplantation methodology. (b) Representative X-ray of the femoral osteotomy and internal fixation of the bone. (c–f) Triple fluorescence images showing signals from DAPI (blue) (c), endogenous RFP (red) (d), anti-Runx2 (green) (e) in the hard callus of fractured cell-injected mice. Overlap between DAPI, RFP, Runx2 is shown in panel f. White inset boxes in the panels show a higher magnification of a representative cell co-expressing DAPI, RFP, and Runx2 signals (white asterisk in the main panels). White arrows indicate RFP^pos Runx2^pos CH cells. (g–j) Triple fluorescence images showing signals from DAPI (blue) (g), endogenous RFP (red) (h), anti-Runx2 (green) (i) in the contralateral femur of fractured cell-injected mice. Overlap between DAPI, RFP, Runx2 is shown in panel (j). Magnification 40X, scale bar 20 μm.

Figure 5. RFP^pos CH cells actively participate in tissue healing.

(a) Serial hystology sections (magnification 5X) from the resulting fracture callus formed 24 days after the osteotomy. #hard callus; °muscle tissue surrounding the callus; *knee region. (b,e,h) Representative anti-RFP immunostaining of the hard callus region derived from fractured and cell-injected mouse (b), corresponding region of the contralateral paw of the same mouse (e), and the hard callus region derived from the fractured and PBS-injected mouse (h). Black arrows indicate osteoblasts within the bone matrix. (c,f,i) Representative anti-RFP immunostaining of the knee region derived from fractured and cell-injected mouse (c), contralateral paw of the same mouse (f), and fractured and PBS-injected mouse (i). Black arrows indicate articular chondrocytes. (d,g,j) Representative anti-RFP immunostaining of the muscle tissue derived from fractured and cell-injected mouse (d), contralateral paw of the same mouse (g), and fractured and PBS-injected mouse (j). Black arrows indicate muscle fibers. Magnification 40X, scale bar 100 μm.