The Treatments of ADSCs in Chronic Stroke
running title: Combined therapy of ADSCs in chronic stroke
Tzu-Min Chan1,2, Horng-Jyh Harn3,4, Hui-Ping Lin5, Shao-Chih Chiu1,13, Po-Cheng Lin6, Hsin-I Wang6, Li-Ing Ho7, Tzyy-Wen Chiou8, An-Cheng Hsieh1, Yu-Wen Chen1, Wen-Yu Ho9,10, Shinn-Zong Lin1,11,12,13＊
1Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan,
R.O.C
2Everfront Biotech Inc., New Taipei City, Taiwan, R.O.C
3Department of Medicine, China Medical University, Taichung, Taiwan, R.O.C 4Department of Pathology, China Medical University Hospital, Taichung, Taiwan,
R.O.C
5Institute of Cellular and System Medicine, National Health Research Institutes,
Miaoli 35053,Taiwan, R.O.C
6Gwo Xi Stem Cell Applied Technology, Hsinchu, Taiwan, R.O.C
7Department of Respiratory Therapy, Taipei Veterans General Hospital, Taipei,
Taiwan, R.O.C
8Department of Life Science and Graduate Institute of Biotechnology, National Dong
Hwa University, Hualien, Taiwan, R.O.C
9Department of Laboratory Medicine, China Medical University Beigan Hospital,
Yunlin, Taiwan, R.O.C
10Department of Medical Technology, Nursing and Management, Jen-Teh Junior
College of Medicine, Taiwan, R.O.C
11Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Taiwan, R.O.C
12Department of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical
University, Tainan, Taiwan, R.O.C
13Graduate Institute of Immunology, China Medical University, Taichung, Taiwan,
R.O.C
＊Address correspondence to Prof. Shinn-Zong Lin, MD., PhD., Center for
Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan, ROC. Tel: +886-4-22052121 ext. 6034; Fax: +886-4-220806666; E-mail:
shinnzong@yahoo.com.tw
keywords: cerebrovascular; chronic stroke; ADSCs; ischemic; hemorrhagic; angiogenesis; angiogenesis; gliosis; neurotrophic factors
ABSTRACT 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Stroke is one of diseases that most needed to find out the clinically effective therapeutic modalities, whereas there are numerous ways attempt to investigate feasibilities. However, inflammatory neuron death would cause irreversible injuries and infarction regions by ischemic or hemorrhagic manner, and thus become a chronic stroke while currently has no drugs to therapy. It is urgent to provide a fundamental treatment method to regenerate brain neuron cells, therefore, use of stem cells for curing chronic stroke would be a major breakthrough development. In this review, we introduced the classification of stroke with its features, as well as focused on the benefits of adipose tissue-derived stem cells and their applications in stroke animal models. The results showed cell-based therapies have significant improvements in neuronal behaviors and functions through different molecular mechanisms, whereas no safety problems arise after transplantation. Further, we proposed a clinical possibility to create a homing niche by reduced invasive intracerebroventricular, and then combined with continues intravenous administration to achieve complete cure.
INTRODUCTION 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
On the basis of World Health Organization statistics, cerebrovascular disease is second only to cancer diseases in top 10 leading causes of death on developing or developed countries (43); thus, cerebrovascular is another intractable diseases. Pathogen of stroke, a most common of cerebrovascular disease, is a blood circulation caused injury and hamper the brain metabolism, whereas eventually lead to impaired brain function (13,47). In clinical diagnosis the majority patients will be able gradually to restore after the acute phase, but brain function has received the irreversible injuries to cause partial loss of nerve function (22). Therefore after a stroke recovering often occur chronic symptoms together with independent capacity loses and paralysis production, resulting in the family and society's burden.
Stroke was mainly due to the blockage of blood vessels in different parts of the brain, resulting different degrees in loss of nerve function with sudden hemiplegia, aphasia, numbness in hands and feet (60,62). Current treatments in the acute phase is to impose anticoagulant, anti-platelet aggregation agents, or thrombolytic agents (12). One of the most commonly used is recombinant tissue plasminogen activator (rt-PA) that is to breakdown blood clots in acute stroke cure (23,51). Only 9% patients being alive within 3 hours of the onset in acute stroke, whereas benefits was not significant over that point in time (71,72). Besides, ischemic or hemorrhagic inflammatory neuronal death, causing irreversible damage and infarct site and thus becoming a 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
chronic stroke. With the applicability of stem cells as well as medical research is widely investigated, medical community began to develop regenerative therapy for chronic stroke (6). Preliminary experimental results indicate that treated patients have significant progress in neurobehavioral, as well as postoperative follow-up period has no abnormal cell proliferation (34,35).
In recent years clinicians expectation that through autologous stem cells to cure chronic stroke, it could prevent carcinogenicity, immune rejection and ethical controversy (13,14). A method has been proposed that G-CSF stimulated proliferation of hematopoietic stem cells that move from marrow to the blood, and then further to separate them via withdrawing blood (62). The results showed autologous stem cell transplanted into the brains are safe, as well as have a fairly good effect in chronic stroke. Nevertheless, the source of hematopoietic stem cells is inadequate, and thus it seems need to search alternative autologous tissues for preparing a large number of stem cells (29). In this review, via literatures examination that there are adequate adipose tissue-derived stem cells (ADSCs) separated from fat tissue with minimal invasive procedures (24,77). In addition, we focused on the efficacy of ADSCs treatment in chronic stroke that explore the mechanisms and effectiveness, as well as carrying out the analysis of future expected human clinical trials.
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
THE APPLICATION OF ADSCs
ADSCs is a kind of mesenchymal stem cells (MSCs) that could be isolated from wide variety of sources with minimally invasive procedures, such as abdominal subcutaneous, intra-articular and visceral depots, as well as ectopic adipose tissues (21). The characteristics of ADSCs are easy to produce large numbers of pluripotent cells from a small amount of tissue as well as in vitro stabilized proliferation without loss stemness, which are essential for regenerative medicine and tissue engineering (2,15). It has been proved that ADSCs expressed the cell surface markers such as CD29, CD44, CD73, CD90, CD105 and HLA class I (76). With defined conditions ADSCs can differentiate into various tissue specific lineages, including adipocytes, osteoblast, hepatocytes, chondrocytes, myocytes, as well as epithelial and neuronal cells (29,66).
For the implementation of clinical trials, the safety issues of ADSCs have been widespread concerned while extensive investigations with its therapeutic capacity in various diseases (54). Different algebra of ADSCs in karyotype analysis (contain chromosomes deletion, translocation, duplication, inversion and insertion) showed no chromosomal mutation occur after twelve in vitro subcultures (53). Comparison of the
p53 gene nucleic acid sequence found no difference between long-term cultured
ADSCs and normal cells (57). For analysis of telomerase activity, it has been proved 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129
the stem cells did not produce telomerase activity scale change within nearly twenty
in vitro generations (57). The most direct evidence is that different algebraic ADSCs
subcutaneously transplanted into nude mice, keeping track for few months, and then found no case to produce abnormal proliferation (58). Moreover, ADSCs has been applied in autoimmune disease, multiple sclerosis, polymyositis, dermatomyositis and rheumatoid arthritis; the results indicate that treated patients have no adverse side effects (53,67). Safety and suitability of the theoretical basis for autologous transplantation, ADSCs has become one of the most popular items in MSCs therapy research and future clinically regenerative medicine.
CLASSIFICATION OF STROKE
Based on the type of blood injury in subventricular zone (SVZ), it could be distinguished into ischemic and hemorrhagic stroke (28,36). Ischemic stroke occurs endovascular occlusion leading to loss of normal blood supply, to cause immediately damage as well as large number of dead nerve cells (55). Moreover, ischemic stroke, accounting 85% of all stroke patients, can be divided into cerebral embolism and thrombosis (1). The main reason was due to atherosclerosis plaque to rupture and thus occur the formation of embolism situation, while high-risk groups are mainly three kind of patients with high incidence (hypertension, high cholesterol and high blood 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148
sugar) (40). Hemorrhagic stroke is commonly known as hyperemia accounted for 15% of all stroke patients, however, it is higher degree of risk than ischemic stroke (65). Vascular brain lesions, congenital (aneurysm) and acquired (hypertension), leading to change blood vessel fragility as well as resulting in increased vascular rupture (19,73).
Regarding to the pathogenesis of stroke that ischemic lesions are relatively comprehensible compared with hemorrhagic symptoms in SVZ (45). The ischemic penumbral region will accelerate the release of glutamate, an excitatory neurotransmitter in central nervous system, to bind and activate N-methyl-D-aspartate (NMDA) receptor (44,45). Activation of NMDA receptors causes large amounts of sodium, calcium, and water flow into the nerve cells, which activate endogenous lysis enzymes to digest the cell components (69,74). Unbalanced flow of calcium ions also result the mitochondria dysfunction, to activate proteins associated with neurological death of nerve cells (46,64). Besides, ischemia-induced free radicals and other reactive oxygen species also can induce cell apoptosis signal (48,64).
Neurological deficit of vascular origin occurs within few minutes to hours that in tendency defined as acute stroke, whereas extensive duration time is chronic stroke (68). Although acute stroke patients could be treated in a hospital with thrombolysis, however, it cause a irreversible damages as well as injured brain (27). Acute therapy 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
behaviors usually only partially restored the wounded area, thus, these injury will evolve as the main problems of chronic stroke. Therefore, clinical treatments of chronic stroke is urgent to restore original nerve cells and their functions in the brain niche.
ADSCs IN CHRONIC STROKE
For treatments in various neurodegenerative diseases, there are many cell or tissue that in vivo transplanted into the human brain, such as aborted fetal tissue containing stem cells (4), ganglion lineage cells (32), umbilical cord blood monocytes (79) and CD34 + hematopoietic stem cells (61). In one case NT2 cell was in vivo transplanted into adult stroke rats, indicating not only restore stroke symptoms but also prolong the survival time (59). The results confirmed that the stem cells has the potential therapy in brain diseases, as well as the safety is no doubts. However, the stem cell used for the treatment of stroke that is not derived from the patients themselves; it need to go through the HLA matching procedures to avoid immune rejection after transplantation (49,61,78). Thus, it is a valuable objective to verify whether the administration of autologous ADSCs into injured penumbral has functional regeneration on the clinical cell-based therapies in chronic stroke (11,24). In fact most of the available reports confirmed successfully administration of ADSCs 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
into injured brains, involving intracerebroventricular and intravenous transplantation for treatment of chronic stroke (Table. 1) (24). Despite all the mechanisms of effective therapy are not well-known, however, it still could classify current evidences as methodical manner
Angiogenesis
Either hemorrhagic or ischemic stroke when it is transformed into chronic type of stroke, injured niche without blood vessels to support nutrient for maintaining cell survival; thus, vascular endothelial growth factor (VEGF) might be a possible mechanism to protects neurons from cell death (18). Immunohistochemical (IHC) staining indicated the proportion of VEGF in treatment group was higher, which inference the secreted VEGF repair brain damage through promote angiogenesis (8). Numerous reports also evidence that ADSCs could restore the function of brain through the secretion of VEGF to promote angiogenesis in injured region (8,26,31).
Neurogenesis
Administration of ADSCs could induce neuronal differentiation, as well as stimulate brain repair markers associated with neurogenesis to functional recovery of chronic stroke (8,24,56). It has been showed that contralateral brain have a small number of newly generate neuron cells, due to a capability of differentiation with 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206
injected ADSCs. IHC staining showed ADSCs differentiate into neuron-like or glialike cells expressing such as neuronal nuclei (NeuN), Nestin and glial fibrillary acidic protein (GFAP) (26,52), which are related with the recovery of brain function (33). Moreover, end of synaptic integration with nerve regeneration involved axonal plasticity and remodeling in brain repair, implying newly derivative cell transform into a functionally integrated neuron (25).
Gliosis
However, it would cause the glial scar in the infarct boundary, when occurs the excess proliferation or hypertrophy of glial cells, such as astrocytes, microglia, and oligodendrocytes (3,10). ADSCs for stroke treatment is to reduce the generation of GFAP+ cells thereby allowing prevent brain nerve fibrosis, which might be one of effective mechanisms (77). Besides, several studies also indicate that ADSCs administration reduces reactive gliosis in response of enhanced neurological outcome, as well as reducing stroke caused infarction region (24,30,37,38).
Apoptosis
Moreover, neuronal recovery accompany with a reduction in brain injury-derived apoptosis as well as natural repair response activated following by brain injury (24). Intravenous administration of ADSCs showed that apoptotic marker (Caspase-3 and 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226
MAP-2) are much lower than the control group, it can be inferred stem cells treatment of stroke by inhibiting apoptosis to achieve a therapeutic effect (77).
Neurotrophic factors
In addition to reducing apoptosis signals, ADSCs also increase neuron cells survival and/or differentiation through neurotrophic factors that may play an important role in maintenance of neural tissue (39,50). A recent report showed that ADSCs can express several neurotrophic factors with improved functional recovery in middle cerebral artery occlusion (MCAO) rats (37). Besides, administration of ADSCs is associated with improvement in stroke deficits by increasing neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) (30,52).
Immune inflammatory response
Another possible mechanism on protective role of ADSCs might due to its immunomodulatory that displayed increased of IL-10 and reduced of TNF-alpha in MCAO rat (16). ADSCs therapy attenuates inflammatory response via reducing the levels of IL-18, TLR-4and PAI-1, to modulate immune reactivity as well as enhancement in neurological outcome (41). In summary, these results indicate neuronal behaviors and functions can get a significant improvement through different 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246
mechanisms after administration of ADSCs in rodent stroke models.
FUTURE EXPECTATION
The main purpose of regenerative medicine studies in chronic stroke is to find out the most effective and safe treatments, which improve functional outcome after brain injury. The neurological recovery of intracerebroventricular administration of ADSCs is greater than intravenous (52), however, invasive delivery might caused intracerebrally trauma and surgical complications. In addition, chronic stroke has not express inflammatory response factors (such as IL-1, TNF-α , etc.), as well as without homing factors (such as SDF-1, CXCR-4, BDNF and GDNF, etc.) to assist in the prognosis of stroke (20,63). According to literatures and our investigations found that ADSCs can secrete homing factor themselves while transplantation into brain niche (5,20). Transplanted ADSCs has in vivo effect of the formation concentration gradient, to attract additional stem cells that administrate from intravenous. It offered a ideal and allowed continues intravenous injection of ADSCs behind a intracerebroventricular cure, to achieve completed therapy with minimal invasive manipulation. Thus, it was an approach worth to apply in clinically for the treatment of chronic stroke with combined therapy of intracerebroventricular and intravenous administrations. 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265
ACKNOWLEDGMENTS
Stem Cell and Regeneration Medicine Foundation, NSC 99-2320-B-039-008-MY3 and Everfront Biotech Inc.
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Table 1. ADSCs application in chronic stroke
Model Animal Dose/Type Protein expression Symptom improvement
ICH (8) rat rADSCs
2-4x105/i.c.v.
VEGF Reduce cell death
Improve neurological function
ICH (75) rat hADSCs
i.v.
Behavioral functional recovery
MCAO (7) rat ADSCs
1x106/ i.c.v.
Neuritin NF200 GFAP ↓
Regeneration of damaged nerve
age-matched C57BL/6J mice (31) mice mADSCs 1X105/i.v. VEGF HGF Angiopoietin-1
Reduce cell death
MCAO (42) rat ADSCs
1x106/ i.c.v.
Bcl-2
Caspase-12 ↓
Reduce cell death Cerebral
ischemia (77)
rat ri-ADSCs Nestin
MAP2 GFAP ↓
Behavioral function improved Reduce brain atrophy
MCAO (16) rat ADSCs
1x106/ i.c.v.
IL-10
TNF-alpha ↓ 547
ICH (38) hADSCs 3X106/i.v.
Reduce cell death Reduce brain atrophy Reduce gliosis
Performance of the endothelial marker
MCAO (37) rat hADSCs
i.c.v.
MAP2 GFAP
Behavioral improvement
MCAO (70) rat hADSCs
1x106/i.c.v.
bFGF VEGF
Enhanced microvessel proliferation ICH; MCAO
(33)
rat hADSCs
40 mg/kg to rats/i.v.
Reduces ischemic region
Ameliorates neurologic deterioration
MCAO (17) mice hADSCs
4×105 /i.c.v.
Reduction in the MCAO-induced infarction volume
MCAO (9) rat hADSCs
1x106/ i.c.v.
VEGF TGF-b1
Increase in the microvessel number Decrease in the neuronal cell apoptosis Neuroprotective
ICH (76) rat hADSCs
i.v.
BDNF GDNF
Behavioral improvement
pMCAO (26) mice rADSCs
2x106/i.v.
VEGF SYP Olig-2
Functional recovery Reductions in cell death
NF oligodendrogenesis, synaptogenesis and angiogenesis
Abbreviation: intracerebral hemorrhage (ICH); middle cerebral artery occlusion (MCAO); intracerebroventricular (i.c.v.); intravenous (i.v) 548
