Distinct Origins of Restricted Progenitors in Postnatal Mouse Blood.

BACKGROUND: Most researchers have accepted that unipotent progenitors are the predominant components in bone marrow for tissue regeneration. However, the unipotent progenitors for blood components are still unclear. We previously found that erythrocytes are derived from a distinct unipotent progenitors, or erythrocyte sacs. METHODS: In the current study, we investigated if the other types of unipotent blood cell progenitors existed, what was their original morphologies, and the mechanism of their generation in mouse blood. RESULTS: We found two morphologically distinct structures that released spore-like small progenitors in mouse blood. One structure was filamentary-like, contained inclusions, widened due to differentiation of the inclusions, and eventually, released spore-like DNA+ and cluster of differentiation 34 (CD34)+ spore-like small progenitors. Another structure was bud-like, contained inclusion, enlarged from less than 10 µm to more than 30 µm, and also released many spore-like small progenitors. Each type of these spore-like progenitors was approximately 1 µm in diameter and could continue to transdifferentiate in circulation. CONCLUSIONS: Our data provide evidence that two types of blood cell restricted progenitors are produced from either filamentary structures or bud-like structures. Both filamentary and bud-like structures were originally released from morphologically distinct, or lineage predetermined tube-shaped structures, or specific niches. Thus, distinct lineages of blood unipotent progenitors are newly produced.

Epithelial stem cells are formed by small-particles released from particle-producing cells.

Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small "naked" nuclei. The small particles were about 2 to 3 µm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some "naked" nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion.

Kidney regeneration by non-platelet RNA-containing particle-derived cells.

We found a group of non-platelet RNA-containing particles (NPRCPs) in human umbilical cord blood. These particles can aggregate, fuse and become non-nucleated cells when cocultured with nucleated cells in vitro. The non-nucleated cells further differentiate into nucleated cells expressing octamer binding transcription factor 4 (OCT4). The NPRCPs are approximately 1-5 µm in diameter, have a thin bilayer membrane, contain short RNAs and microRNAs and express OCT4, sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). To confirm the function of NPRCPs in vivo, we examined the effects of tail vein-injected green fluorescent protein (GFP)-labelled NPRCPs on mouse kidneys damaged by prior ischaemia and reperfusion from Day 1 to Week 6. Within 1 day of injection of NPRCPs, immunofluorescence and immunohistochemistry revealed a large number of extravasated NPRCPs in the renal calyces, damaged glomeruli and duct tubules. During the course of regeneration, NPRCPs fused into large, non-nucleated cellular structures that further became large nucleated cells to regenerate multicellular kidney tubules. In addition, many NPRCPs became tiny nucleated cellular structures that further differentiated into interstitial cells in connective tissue. The extravasated NPRCPs also arranged themselves into non-cell glomerular structures before further regenerating into nucleated cells of the glomerulus. In conclusion, the results demonstrate that, via different patterns of differentiation, NPRCP-derived cells can regenerate mouse kidney tissue damaged by ischaemia.

Pre-stem cell formation by non-platelet RNA-containing particle fusion.

We found a group of non-platelet RNA-containing particles (NPRCP) in human umbilical cord blood. To understand the origin, characterization and differentiation of NPRCP, we examined cord blood-isolated NPRCP in vitro. The NPRCP range in size from < 1 to 5 µm, have a thin bilayer membrane and various morphological features, contain short RNA and microRNA and express octamer-binding transcription factor 4 (OCT4), sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). On coculture with nucleated cells from umbilical cord blood, NPRCP fuse to small, active, non-nucleated cells called 'particle fusion-derived non-nucleated cells' (PFDNC). The PFDNC are approximately 8 µm in diameter and are characterized by their twisting movement in culture plates. They can easily move into and out of nucleated cells and finally differentiate into mesenchymal-like cells. In addition, the larger non-nucleated cellular structures that are derived from the aggregation and fusion of multiple NPRCP can further differentiate into large stem cells that also release OCT4- and SOX2-positive non-nucleated small cells. Our data provide strong evidence that NPRCP can fuse into PFDNC, which further differentiate into mesenchymal-like cells. Multiple NPRCP also fuse into other types of large stem cells. We believe that stem cells are derived from NPRCP fusion. There is considerable potential for the use of NPRCP in clinical therapy.

Germ plasm-like Dot cells maintain their wound regenerative function after in vitro expansion.

1. Wounds in fetal skin heal without scarring; however, the mechanism for this is unknown. We have identified a novel group of protein and nucleotides-positive particles in fetal and adult mouse blood and in human blood, and termed them 'Dot cells'. Freshly isolated Dot cells regenerate wounds with less scarring and can be cultured without feeder layers. 2. Because the morphology of Dot cells has never been described, in the present study we describe the specific characterizations of Dot cells, including their growth pattern in vitro, and their expressions of stem cell markers using fluorescent cell sorting analyses and immunofluorescent histology. Our data indicates that cultured Dot cells express stem cell surface markers and embryonic stem cell transcription markers, such as Oct4, Nanog and Sox-2. In addition, Dot cells express VASA, the germ plasm specific marker. 3. To confirm whether Dot cells maintain their wound regenerative activity after in vitro expansion, in vitro cultured Dot cells were transplanted to wounded mice. Dot cells from albino mice maintain their wound regenerative activities after intravenous transplantation to black-background diabetic mice. In addition, Dot cells regenerate both the epithelial and dermal cells in the wounds of wild-type mice. The regenerated hair follicles, smooth muscle and dermal tissues express transiently to VASA. 4. Our data demonstrate that Dot cells are newly identified organisms located in the blood and bone marrow of mammals. They express germ cell, embryonic stem cell and adult stem cell markers. Dot cells maintain their regenerative function after in vitro expansion.

Upregulation of neurodevelopmental genes during scarless healing.

Scarless fetal skin wound healing is a paradigm for ideal skin repair and is dependent on peripheral nerve function.To further explore neurogenic mechanisms influence on the scarless skin repair, fetal rats were wounded on gestational days 16 (E16; n = 24) and 18 (E18; n = 8) and wounds were harvested at 1 and 3 days after injury. Unwounded skin at identical gestational age was used for control comparison. The scarless E16 and scarring E18 wounds underwent macroarray gene expression analysis (1172 genes).During the scarless healing period, 53 (4.5%) genes had a statistically significant upregulation post-injury with at least a 2- to 3-fold change 1 day after wounding and 14 (1.2%) genes 3 days after wounding (P < 0.05). Many neurodevelopmental genes were increased during scarless repair on post-injury days 1 and 3. Neuropeptide Y Receptor type I, cJun related Transcription Factor (junD), Synaptophysin, SNAP 25, Neuronal calcium sensor 1 (NCS1), neural visine-like calcium binding protein 1 (NVP1), nerve growth factor-induced gene A (NGFI-A/EGR1), VGF8A protein, p27kip1, and members of the GABA and serotonin family each had 2- to 3-fold expression increases (P < 0.05).We speculate that fetal skin cells express neurotrophins during skin development that regulate peripheral neuron formation. During injury these factors promote the survival and regeneration of peripheral neurons; this interaction of neuropeptides, neuropeptide receptors, and neurotrophins may modulate the fetal scarless repair mechanisms in response to injury. Identification of these neurodevelopmental candidate genes provides insight for new investigation into mechanisms regulating scarless healing.

Interaction of wingless protein (Wnt), transforming growth factor-beta1, and hyaluronan production in fetal and postnatal fibroblasts.

BACKGROUND: Mammalian fetal skin injury heals scarlessly. The intrinsic differences between embryonic and adult fibroblasts that underlie this observation are poorly understood. Several studies have linked Wnt proteins with skin morphogenesis. The authors' study aimed to establish a correlation between beta-catenin-dependent (canonical) Wnt protein, transforming growth factor (TGF)-beta1, and the expression of hyaluronan synthesis enzymes during scarless versus scarring wound healing. METHODS: Wnt signaling was quantified after 1.5-mm skin wounds were created in BAT-gal fetal (e16.5) and postnatal (p1) mice. Canonical Wnt signals were localized by X-gal staining and quantified with quantitative real-time polymerase chain reaction. Primary embryonic and postnatal mouse dermal fibroblasts were treated with recombinant Wnt3a or TGF-beta1. Proliferation was assayed by bromodeoxyuridine incorporation. Gene expression of enzymes that regulate hyaluronan production and turnover was examined by quantitative real-time polymerase chain reaction (hyaluronan synthases or HAS1-3, hyaluronadase-2), as well as other target genes for Wnt and TGF-beta (Axin2, TGF-beta1, TGF-beta3, type 1 collagen, proliferating cell nuclear antigen). RESULTS: Canonical Wnt signaling increased following wounding in postnatal, but not fetal, mice. In vitro, rmWnt3a increased postnatal fibroblast proliferation but not in embryonic cells. Both Wnt3a and TGF-beta1 induced HAS2 and HAS3 gene expression in embryonic fibroblasts, while HAS1 and Hyal2 were induced in postnatal fibroblasts. Finally, rmWnt3a significantly increased type I collagen expression, particularly in postnatal fibroblasts, and influenced expression of TGF-beta isoforms. CONCLUSIONS: Increased canonical Wnt signaling occurs during postnatal but not fetal cutaneous wound repair. Fetal and postnatal fibroblasts have a disparate response to rmWnt3a in vitro. rmWnt3a affects postnatal fibroblasts in a similar fashion to rhTGF-beta1, a known profibrotic cytokine.

Cyclophilin C-associated protein/Mac-2 binding protein colocalizes with calnexin and regulates the expression of tissue transglutaminase.

Cyclophilin C-associated protein (CyCAP) or Mac-2 binding protein has been identified as a binding protein for cyclophilin C in mice and for Mac-2 (galectin-3) in human, suggesting its multiple binding activity to proteins. In the present study, using specific anti-rat-CyCAP antibody, we found that CyCAP colocalizes with calnexin at the location near the nuclear envelope, however CyCAP does not have colocalization with calreticulin. In senescent fibroblasts and interferon-gamma (IFNgamma) treated fibroblasts, both calnexin and CyCAP form larger polymers and are released from the endoplasmic reticulum (ER) through the cellular membrane to the extracellular area. Immunoprecipitation studies further confirm that the release of calnexin is through binding to CyCAP. Further, we found that tissue transglutaminase (tTG) protein is decreased, however not at the RNA level, in CyCAP null fibroblasts, which suggests that CyCAP is involved in tTG post-translational modification. Our data give novel evidence that CyCAP regulates the post-translational modification of tTG through its colocalization with calnexin in ER.

Blood-derived small Dot cells reduce scar in wound healing.

Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.

Increased CCN2 transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites.

OBJECTIVE: We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth. SUMMARY BACKGROUND DATA: Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal. METHODS: Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers. RESULTS: Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun. CONCLUSIONS: We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

Cyclophilin C-associated protein is up-regulated during wound healing.

Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.

Epithelial-mesenchymal transition occurs after epidermal development in mouse skin.

In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.

Skin wounds in the MRL/MPJ mouse heal with scar.

Adult MRL/MpJ mice regenerate cartilage during repair of through-and-through ear punch wounds. However, the ability of this mouse strain to heal isolated cutaneous wounds by regeneration or with scar is unknown. The purpose of this study was to characterize the rate of reepithelialization and collagen architecture in dermal wounds from MRL/MpJ mice compared with C57bl/6 and Balb/c strains. Full-thickness incisional (5 mm) and excisional (2 mm diameter) skin wounds were made on the dorsum of 7-week-old MRL/MpJ, C57bl/6, and Balb/c mice. Ear punch wounds were made simultaneously on each animal. Reepithelialization was complete by 48 hours for incisional skin wounds in each strain. All excisional wounds showed incomplete reepithelialization at 24, 48, and 72 hours. At 14 days, all skin wounds had grossly healed. In contrast to the ear wounds made in C57bl/6 and Balb/c mice, MRL/MpJ ear wounds were completely healed by day 28. Dorsal skin wound sections at 14 and 28 days revealed dense collagen deposition and similar degrees of fibrosis between the three strains of mice. In conclusion, in contrast to wound healing in the ear, MRL/MpJ mouse dorsal cutaneous wounds heal similarly to C57bl/6 and Balb/c mice with dermal collagen deposition and scar formation.

Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation.

BACKGROUND: Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation. METHODS: Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression. RESULTS: Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively). CONCLUSIONS: Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

Cyclophilin C-associated protein is a mediator for fibronectin fragment-induced matrix metalloproteinase-13 expression.

The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

Molecular cloning and expression of keratinocyte proline-rich protein, a novel squamous epithelial marker isolated during skin development.

We describe a novel rat cDNA named keratinocyte proline-rich protein (KPRP) isolated by RNA differential display during skin development. We determine that KPRP is expressed in stratified squamous epithelium, and its approximately 2.8-kb cDNA encodes a 699-amino acid protein with high proline content (19%). KPRP is an insoluble protein, similar to most epidermal terminal differentiation-associated proteins. Immunoblot of the protein lysate from keratinocytes, using strong reducing conditions, demonstrates two KPRP bands of approximately 76 and 55 kDa size. KPRP is expressed in stratified squamous epithelia of skin, tongue, and esophagus. The initiation of KPRP expression in fetal rat skin at E17, E18, E19, E20, and E21 was analyzed by reverse transcription-PCR. Fetal skin at E19 and later expresses KPRP. In situ hybridization of skin from E18, E19, and 4-day-old neonatal rats demonstrates that interfollicular and follicular keratinocytes express KPRP. Anti-KPRP antibody demonstrates KPRP protein localizes to all layers of stratified epithelia in skin, tongue, and esophagus. In cultured dermal keratinocytes, KPRP is diffusely distributed throughout the cytoplasm with denser staining adjacent to the nuclear and plasma membranes. Additionally, immunoreactive intracellular granules are observed during keratinocyte detachment from their plastic substrate. Rat KPRP has 89% homology to a mouse genomic DNA sequence and 56% homology to a human hypothetical protein. We conclude that KPRP may be a new epidermal terminal differentiation-related protein expressed in stratified squamous epithelia. KPRP is expressed by fetal dermal keratinocytes during late gestation and is a new marker of maturing epidermis during fetal skin development.

Proteinase-Activated Receptors: New Functions for Old Enzymes.

Although proteases are traditionally viewed as degradative enzymes, characterization of a family of G protein-coupled receptors that are activated by proteolysis reveals a new role. Certain proteases function as signaling molecules that specifically regulate cells by cleaving and activating a family of proteinase-activated receptors.