**2. Wound repair and innate and acquired immunity**

#### **2.1. Skin wound healing**

Wound healing is one of the most important subjects in medicine and biology. For example accidental injuries or medical surgery initiate wound repair processes. From the practical and fundamental importance wound healing has been the subject of intense investigation for many years [2]. Recently increase of elderly person necessitates the research of wound repairfurther. The ability to wound repair declines with age, which induces incurable pressure ulcer in elderly patients [3]. Acute inflammatory reactions play integralroles in normal wound healing and tissue repair. Once parenchyma and blood vessel are damaged, the coagulation system is activated, which release chemical mediators that promote vascular permeability and leukocyte adhesion and recruitment. Within 30 min. neutrophils migrate to the site of wound, which phagocytize dead cells and cellular debris and clear tissue. Then neutrophils are themselves phagocytized by tissue macrophages. Although M1 macrophages damage tissue cells pro‐ ducing tissue‐damaging proteases, which are primarily kill microorganisms, M1 macrophages also engulf damaged tissue cells. Then M2 macrophages migrate from blood vessel to repair tissue. Neutrophils have specific cell surface antigens called, Gr‐1. In murine model of wound repair (Figure 2), we depleted neutrophils by anti‐GR‐1 antibody. Although in young mice the kinetics of wound healing was not different by the depletion of neutrophils, the depletion of neutrophils by anti‐Gr‐1 antibody dramatically delayed wound healing in aged mice [4]. Bone marrow has both matured neutrophils and macrophages. First, we found that in splenectom‐ ized mice, bone marrow, spleen and thymus injection accerated wound healing (Figure 3). We transplanted bone marrow cells from GFP mice (C57BL/6 background) to C57BL/6 mice. We found that bone marrow cells injection accelerated wound healing and GFP positive neutro‐ phils and macrophages migrated to the wound tissue. Because wound healing of aged mice is relatively inefficient, we transplanted young bone marrow cells to aged mice. We found that young bone marrow cells enhanced wound healing of aged mice [5, 6]. In case of microorgan‐ isms acquired immunity help to phagocytize invader. By microorganism's infections, immu‐ noglobulin specific for the microorganisms bind to the surface and induce phagocytosis mediated by Fc receptors.We examined whether B cells, which produce antibodies to damaged tissues, might be engaged in the process of wound healing. We found that wound healing in splenectomized nude mice was delayed and the transfer of B cells accelerated wound healing in splenectomized mice. Further we detected several autoantibodies binding to wounded tissues [7] (Fig.4). Advancing age gradually decreased the strength of IgG1 autoantibodies, which bind to wounded tissues, although the strength of IgM autoantibodies was relatively stable by advancing age [5, 6].

#### **2.2. DSS‐induced colitis**

Microorganisms External stimuli

damages

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

DAMPs

Macrophages Dendritic cells

PRRs

Pro-inflammatory cytokines

**Figure 1.** Damaged cells by various stimuli produce DAMPs, which activate tissue resident macrophages and dendritic

Wound healing is one of the most important subjects in medicine and biology. For example accidental injuries or medical surgery initiate wound repair processes. From the practical and fundamental importance wound healing has been the subject of intense investigation for many years [2]. Recently increase of elderly person necessitates the research of wound repairfurther. The ability to wound repair declines with age, which induces incurable pressure ulcer in elderly patients [3]. Acute inflammatory reactions play integralroles in normal wound healing and tissue repair. Once parenchyma and blood vessel are damaged, the coagulation system is activated, which release chemical mediators that promote vascular permeability and leukocyte

cells. Activated innate cells produce pro-inflammatory cytokines.

**2.1. Skin wound healing**

Biomedical Engineering

208

**2. Wound repair and innate and acquired immunity**

Internal stimuli

As a mucosal wound repair model we used dextran sulfate sodium (DSS). Colitis may result from DSS toxicity to colonic epithelial cells. DSS is commonly used in rodent models to chemically induce acute intestinal inflammation, and the DSS‐induced colitis is characterized by weight loss, bloody diarrhea, epithelial cell damage, and immune cell infiltration, as well as an increased production of inflammatory mediators including TNF‐α, IL‐6, IL‐12, and interferons. Colitis was induced by oral administration of DSS to two months old C57BL/6 mice at 2 % (w/v)in drinking water ad libitum forfive days followed by normal drinking water. By this schedule, mice almost completely recovered. Interestingly at recovery phase of DSS‐ induced colitis, we observed strong up‐regulation of innate immune cells having Gr1+ CD11b + cell surface maker in spleen and bone marrow. Transplantation of splenic DSS‐derived Gr1+ CD11b+ cells into DSS‐treated mice improved colitis and promoted efficient colonic mucosal healing [8] (Figure 5). Anti‐Gr‐1 antibody treatment worsened the DSS‐ administered colitis. These results indicate that Gr1+CD11b+ cells induced by DSS worked to repair colon wound healing and repair colitis [9].

250 KDa 150 KDa 100 KDa 75 KDa

*< Immunoprecipitation >*

Sham Splenectomy

**226KDa** Myosin, heavy polypeptide 9, non-muscle isoform 1

Day 11 after DSS

**164KDa** Carbamoyl-phosphate synthase [ammonia], mitochondrial precursor

**42KDa** Actin, alpha cardiac muscle

Tissue Damage and Repair Caused by Immune System and Personalized Therapy of Failed Organs by Stem Cells 211

*B6 mice (2.5M)*

**Figure 4.** Immunoprecipitation of autoantibodies, which were bound to tissue cells. After separation each band was

Control

DSS only DSS+ GR-1+CD11+ transplantation

**Figure 5.** Colitis was induced by oral administration of DSSat 2 % (w/v) in drinking water ad libitum for five days fol‐ lowed by normal drinking water. Upper show the FACS analysis of spleen of DSS-treated mice. Increased GR-1+CD11b

+ cells were transplanted to mice of at the last day of DSS administration.

50 KDa

37 KDa

25 KDa

analyzed by LC/MS.

Day 5 after DSS treatment

**Figure 2.** Murine experimental model of wound healing. The dorsal skin was punched through with a sterile disposa‐ ble biopsy punch (diameter 3 mm). Separated immune cells were injected intravenously. The wounds and their sur‐ rounding areas were cut for analyses with biopsy punch with a diameter of 6 mm at each day after wounding.

**Figure 3.** Examples of analysis. After 3 weeks of the removal of spleen, bone marrow, spleen or thymus cells were injected at the same time of punch biopsy.

*Experimental design*

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

control

**Figure 2.** Murine experimental model of wound healing. The dorsal skin was punched through with a sterile disposa‐ ble biopsy punch (diameter 3 mm). Separated immune cells were injected intravenously. The wounds and their sur‐ rounding areas were cut for analyses with biopsy punch with a diameter of 6 mm at each day after wounding.

> *Immune cell injection ~ immune cells ~*

**Figure 3.** Examples of analysis. After 3 weeks of the removal of spleen, bone marrow, spleen or thymus cells were

Control 24 h 48 h 96 h

Skin wounds by 3mm punch biopsy

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210

Take samples by 6mm punch biopsy

PBS

BM

Spleen

Thymus

injected at the same time of punch biopsy.

*I.V.* injection of immune cells

FACS analysis

> > 0h 24h 48h 96h

PBS Spleen Bone Marrow Thymus

Sample preparation

**Figure 4.** Immunoprecipitation of autoantibodies, which were bound to tissue cells. After separation each band was analyzed by LC/MS.

**Figure 5.** Colitis was induced by oral administration of DSSat 2 % (w/v) in drinking water ad libitum for five days fol‐ lowed by normal drinking water. Upper show the FACS analysis of spleen of DSS-treated mice. Increased GR-1+CD11b + cells were transplanted to mice of at the last day of DSS administration.

produced matrix metalloproteinase (MMP3, MMP12 and MMP13), which might degrade damaged tissue further or might work to degrade Aβ to protect against dangerous stimuli [18]. Aβ induced microglial migration may induces cytoskeletal system. We have shown that Aβ binds to IQGAP1 and actin, which are cytoskeletal components, and may stimulate Rho/Rac

Tissue Damage and Repair Caused by Immune System and Personalized Therapy of Failed Organs by Stem Cells 213

D‐gal at high levels induces the production of reactive oxygen species (ROS) and advanced glycation end products (AGEs; [20]) and induces sterile inflammation. We administered 50 mg D‐gal for 60 days to 2 months old mice. We found unorganized distributions of keratin‐5 and keratin‐8 proteins in the thymus of these hosts, which resembled to aged mice [21].

Our body has the capacity to regenerate from injury. However, damaged tissues induced by continuous microbial or sterile stimuli cannot be recovered. Continuous stimulation by TGFβ produced by macrophages induces fibroblasts to secrete matrix proteins such as fibrinogen. Damaged tissue cells are replaced by fibrous component. Many patients are suffering from these conditions. About 90% of all deaths from chronic obstructive lung diseases (COPD) are attributable to cigarette smoking. Long term Cigarette smoking induces continuous sterile inflammation. Patients who have low blood oxygen levels in their blood are given supplemental oxygen. However, oxygen supply cannot resume lung function. Long‐term heavy alcoholic stimuli induce continuous sterile inflammation in liver and eventually develop cirrhosis. Adult onset diabetes mellitus is now found to be caused by repeated sterile inflammation, which is derived from obesity. Renal failure has numerous causes. The most common is diabetes mellitus. Lung, liver, and kidney failure has no treatment to cure diseases except transplantation. Transplantation to these diseas‐ es has limitation from organ shortage and immune rejection caused by HLA discrepancy. Another common degenerative diseases are Alzheimer and Parkinson disease. These diseases also are caused by continuous sterile inflammation. Because these diseases affect single organ, we may use healthy other tissue cells for regenerative medicine. Several trials

**4.2. Muscle regeneration from pluripotent Embryonic Stem (ES) cells**

Aged person, who has long been in bed, has serious problem of muscle atrophy. We set up murine model of muscle regeneration. ES cells having LacZ marker were cultured on Type IV collagen dishes with 10% FCS. After 5 days of culture PDGFR‐α<sup>+</sup> mesodermal progenitor population was sorted and transplanted to injured muscle of KSN nude mice. We observed LacZ positive CD34+ Pax7+ cells (satellite cells) in transplanted muscle. Because of transplant‐

signaling [19].

**4.1. Overview**

are now ongoing [22].

**3.4. D‐galactose (D‐gal) induced thymus degeneration**

**4. Toward personalized cell therapy**
