**4. Toward personalized cell therapy**

#### **4.1. Overview**

**3. Sterile inflammation to tissue degeneration**

Neutrophils and macrophages produce pro‐inflammatory cytokines, including interleukins 1 alpha and beta (IL‐1α and β) and tumor necrosis factor alpha (TNF‐α), chemokines, matrix proteases. These materials help to destroy invading bacteria and virus. However, invaded tissue also be damaged by these inflammatory reactions. Once microorganisms are cleared by immune system, the invaded tissue is repaired to healthy state. However, some microorgan‐ isms such as hepatitis C virus keep staying in host tissue and repeatedly attack host tissue, which induces fatal tissue damages finally. In sterile inflammation similar phenomena occur. External stimuli, which destroy tissue cells, produce DAMPs and induce sterile inflammation. Because immune cells secrete toxic proteases to kill microorganisms, the same immune cells destroy tissue cells.If external stimuli are limited,repair system such as M2 macrophages work to repair tissue damages and returns to normal condition. However, repeated stimuli may induce next inflammation before tissue repair and will induce chronic tissue damages, which

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

cannot be recovered. In these processes, auto reactive T cells and B cells are engaged.

**3.2. Bleomycin (BLM)‐induced murine model of experimental systemic sclerosis**

BLM is a glycopeptide produced by the bacterium Streptomyces verticillus, which can cleave DNA, and it is widely used as an anti‐tumor agent for various types of malignancies [10]. Thus BLM administration destroys tissue cells and induces sterile inflammation. We examined the effects of repeated injection of BLM to adult mice. When BLM was injected into the shaved backs of adult C3H or BALB/c mice (100 μg/mouse) 5 days per week for 3 weeks, not only skin fibrosis, but also esophageal and gastric damage related to fibrosis were observed. Injection of BLM induced innate immune inflammation, which did not resolve. Dendritic cells, which engulfed dead cells, transferred auto antigens to T cells. Transfer of CD4+ T cells from BLM‐ treated BALB/c mice induced the same pathological changes and antibody production in untreated‐BALB/c nude mice. Th17 cells secret IL17, which stimulated innate immune cells to

Aβ accumulation is thought to be central to the pathogenesis of Alzheimerʹs disease (AD) [12]. Aβ is produced by proteolysis processing of Amyloid precursor protein (APP). A balance between amyloidogenic processing of APP and the removal of soluble Aβ by clearance pathways and enzyme‐mediated degradation maintains Aβ levels. Aβ is now considered to be one of DAMPs, which activate innate immune cells especially microglia. Putative sensors of Aβ are NLRP3, CD36 andRAGE [13‐15]. By using microglial cells or cell line, we have shown that Aβ produce several cytokines including M‐CSF by PI3K/AKT and NFκB pathway, which stimulate microglial proliferation and migration [16]. Further Aβ induces several chemokines (CCL7, CCL2, CCL3, CCL4 and CXCL2) by PI3K/AKT and Erk pathway in the microglia, which induce the migration of microglia or macrophages [17]. Aβ induced sterile inflammation also

**3.1. Overview**

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damage tissues [11].

**3.3. Amyloid‐beta peptide (Aβ) works as a DAMP**

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 are now ongoing [22].

#### **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‐

ed cells are satellite cells; they may autonomously proliferate *in vivo* [23]. Then we tried to differentiate skeletal muscle cells without serum. We cultured ES cells with BMP4 without serum. By this condition ES cells differentiated into osteochondrogenic cells *in vitro* and *in vivo*. Early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells and finally differentiated to skeletal muscle cells *in vitro* [24].

**4.4. Establishment of induced Pluripotent Stem Cells (iPSCs) from aged mice**

drop followed by two‐dimensional culture [26].

Aged C57BL/6 with GFP

with Flk‐1+ cells [27].

The generation of induced pluripotent stem cells (iPSCs) from murine and human somatic cells opens the possibility of personalized cell therapies for treating human disease and/or repairing the damaged tissues of elderly patients [25]. By the lifespan extension many developed countries has large number of elderly patients, who suffer from incurable diseases. For personalized celltherapy of elderly person,technology to establish iPSCs from aged person is necessary. FurtheriPSCs from aged person need to differentiate to target tissue cells.In order to establish model system to treat elderly patient by iPSCs, we designed murine model. We found that efficiency to establish iPSCs from aged mice was low. However, we could succeed to establish 2 clones of iPSCs from bone marrow derived dendritic cells of 21 month old C57BL/ 6 mice carrying GFP marker by retrovirus encoding fourfactors (Oct3/4, Sox2, Klf4 and c‐Myc). Established iPSCs have pluripotent makers such as SSEA‐1, Oct‐4 and Pou5f1 and alkaline phosphatase activity was positive. Our Aged‐iPSCs made teratoma‐contained tissues of three germ layers, when they were transplanted to the dorsal flank of C57BL/6 (syngeneic) mice. They also differentiate to various tissue cells including muscle, liver and neuron by hanging

Tissue Damage and Repair Caused by Immune System and Personalized Therapy of Failed Organs by Stem Cells 215

By using iPSCs we tried to differentiate into cardiovascular cells for the treatment of ischemic tissue, which might be caused by atherosclerosis. We simply cultured iPSCs in Type IV collagen dish and separated Flk1+ cells by magnetic‐activated cell sorting (MACS) or FACS sorting. Flk1+ cells from iPSCs co‐cultured with HUVECs on Matrigel made the network structures. Flk1+ cells were transplanted adductor muscles in the ischemic limb of nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted

> Chimera formation

**Figure 7.** Establishment of aged-iPSC from GFP positive C57BL/6 mice. Chimera mice production from these iPSCs.

iPSCs

### **4.3. Thymic epithelial cells from induced pluripotent stem cells**

One of the most serious problems of aged person is infection caused by age‐associated immune functional decline. The age‐associated atrophy of the thymus and the decline of naïve T cells output are mainly caused by degeneration of thymic epithelial cells (TEC). We tried to differentiate iPSCs to TEC. The thymus initially develops as an endodermal epithelial cell at embryonic day 10 encapsulated by mesenchyme. We follow this embryon‐ ic development to induce TEC from iPSCs. First, we try to differentiate iPSCs to mesendo‐ derm to definitive endoderm. We added activin A and Lithium chloride (LiCl) to iPSCs in collagen IV –coated dishes. We found that endodermal epithelial cell cluster were sur‐ rounded by mesoderm cells same as embryonic development. By adding FgF8 then FgF7 and FgF10, the population of endodermal epithelial cells enlarged and matured to TEC progenitor. By the addition of RANK ligand TEC progenitor differentiated to medullary TEC like cells.

**Figure 6.** Upper figure shows the fetal development of TEC. Lower figure shows the protocol of TEC differentiation from iPSCs.

### **4.4. Establishment of induced Pluripotent Stem Cells (iPSCs) from aged mice**

ed cells are satellite cells; they may autonomously proliferate *in vivo* [23]. Then we tried to differentiate skeletal muscle cells without serum. We cultured ES cells with BMP4 without serum. By this condition ES cells differentiated into osteochondrogenic cells *in vitro* and *in vivo*. Early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells and finally differentiated to skeletal muscle cells *in vitro* [24].

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

One of the most serious problems of aged person is infection caused by age‐associated immune functional decline. The age‐associated atrophy of the thymus and the decline of naïve T cells output are mainly caused by degeneration of thymic epithelial cells (TEC). We tried to differentiate iPSCs to TEC. The thymus initially develops as an endodermal epithelial cell at embryonic day 10 encapsulated by mesenchyme. We follow this embryon‐ ic development to induce TEC from iPSCs. First, we try to differentiate iPSCs to mesendo‐ derm to definitive endoderm. We added activin A and Lithium chloride (LiCl) to iPSCs in collagen IV –coated dishes. We found that endodermal epithelial cell cluster were sur‐ rounded by mesoderm cells same as embryonic development. By adding FgF8 then FgF7 and FgF10, the population of endodermal epithelial cells enlarged and matured to TEC progenitor. By the addition of RANK ligand TEC progenitor differentiated to medullary

**4.3. Thymic epithelial cells from induced pluripotent stem cells**

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Biomedical Engineering

214

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The generation of induced pluripotent stem cells (iPSCs) from murine and human somatic cells opens the possibility of personalized cell therapies for treating human disease and/or repairing the damaged tissues of elderly patients [25]. By the lifespan extension many developed countries has large number of elderly patients, who suffer from incurable diseases. For personalized celltherapy of elderly person,technology to establish iPSCs from aged person is necessary. FurtheriPSCs from aged person need to differentiate to target tissue cells.In order to establish model system to treat elderly patient by iPSCs, we designed murine model. We found that efficiency to establish iPSCs from aged mice was low. However, we could succeed to establish 2 clones of iPSCs from bone marrow derived dendritic cells of 21 month old C57BL/ 6 mice carrying GFP marker by retrovirus encoding fourfactors (Oct3/4, Sox2, Klf4 and c‐Myc). Established iPSCs have pluripotent makers such as SSEA‐1, Oct‐4 and Pou5f1 and alkaline phosphatase activity was positive. Our Aged‐iPSCs made teratoma‐contained tissues of three germ layers, when they were transplanted to the dorsal flank of C57BL/6 (syngeneic) mice. They also differentiate to various tissue cells including muscle, liver and neuron by hanging drop followed by two‐dimensional culture [26].

By using iPSCs we tried to differentiate into cardiovascular cells for the treatment of ischemic tissue, which might be caused by atherosclerosis. We simply cultured iPSCs in Type IV collagen dish and separated Flk1+ cells by magnetic‐activated cell sorting (MACS) or FACS sorting. Flk1+ cells from iPSCs co‐cultured with HUVECs on Matrigel made the network structures. Flk1+ cells were transplanted adductor muscles in the ischemic limb of nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk‐1+ cells [27].

**Figure 7.** Establishment of aged-iPSC from GFP positive C57BL/6 mice. Chimera mice production from these iPSCs.
