Health Span-Extending Activity of Human Amniotic Membrane- and Adipose Tissue-Derived Stem Cells in F344 Rats

Abstract

Aging brings about the progressive decline in cognitive function and physical activity, along with losses of stem cell population and function. Although transplantation of muscle-derived stem/progenitor cells extended the health span and life span of progeria mice, such effects in normal animals were not confirmed. Human amniotic membrane-derived mesenchymal stem cells (AMMSCs) or adipose tissue-derived mesenchymal stem cells (ADMSCs) (1×10(6) cells per rat) were intravenously transplanted to 10-month-old male F344 rats once a month throughout their lives. Transplantation of AMMSCs and ADMSCs improved cognitive and physical functions of naturally aging rats, extending life span by 23.4% and 31.3%, respectively. The stem cell therapy increased the concentration of acetylcholine and recovered neurotrophic factors in the brain and muscles, leading to restoration of microtubule-associated protein 2, cholinergic and dopaminergic nervous systems, microvessels, muscle mass, and antioxidative capacity. The results indicate that repeated transplantation of AMMSCs and ADMSCs elongate both health span and life span, which could be a starting point for antiaging or rejuvenation effects of allogeneic or autologous stem cells with minimum immune rejection.

Significance: This study demonstrates that repeated treatment with stem cells in normal animals has antiaging potential, extending health span and life span. Because antiaging and prolonged life span are issues currently of interest, these results are significant for readers and investigators.

Keywords: Adipose tissue-derived mesenchymal stem cell; Amniotic membrane-derived mesenchymal stem cell; Cognitive function; Health span; Life span; Physical activity.

Figure 1.

Stem cell transplantation recovers physical activity and cognitive function. (A–C): Locomotor activity (resting, slow-moving, and fast-moving times) (A), rotarod latency (B), and maximum swimming time (C) of rats at 11–20 months of age. (D): Retention time and escape latency in passive avoidance (left) and water-maze (right) performances at 21 months of age. White bar, young rats (7 weeks old); black bar, aged rats; striped bar, aged rats transplanted with AMMSCs (10⁶ cells); checked bar, aged rats transplanted with ADMSCs (10⁶ cells). ∗, significantly different from young rats (p < .05). #, significantly different from aged rats (p < .05). Abbreviations: ADMSC, adipose tissue-derived mesenchymal stem cell; AMMSC, amniotic membrane-derived mesenchymal stem cell; mon, months; sec, seconds.

Figure 2.

Stem cell transplantation recovers acetylcholine concentration and extends life spans of aged rats. (A): Acetylcholine concentration in CSF and muscles. (B): Survival rate (left) and mean life span (right). White bar, young rats (7 weeks old); triangle symbol and black bar, aged rats; square symbol and striped bar, aged rats transplanted with AMMSCs (10⁶ cells); diamond symbol and checked bar, aged rats transplanted with ADMSCs (10⁶ cells). ∗, significantly different from young rats (p < .05). #, significantly different from aged rats (p < .05). Abbreviations: ADMSC, adipose tissue-derived mesenchymal stem cells; AMMSC, amniotic membrane-derived mesenchymal stem cells; CSF, cerebrospinal fluid; mon, months.

Figure 3.

Transplanted stem cells differentiate into neurons, producing ChAT protein. (A, B): Distribution and differentiation of transplanted human (hMito-positive) amniotic membrane-derived mesenchymal stem cells (A) and adipose tissue-derived mesenchymal stem cells (B) into neurons (NF-H- and MAP2-positive) and cholinergic neurons (ChAT-positive). Scale bar = 20 μm. Abbreviations: ChAT, choline acetyltransferase; hMito, human mitochondria; MAP2, microtubule-associated protein 2; NF-H, neurofilament-high molecular weight protein.

Figure 4.

Transplanted stem cells restore cholinergic and dopaminergic systems, and increase growth and neurotrophic factors. (A, B): Polymerase chain reaction analysis of cholinergic (A) and dopaminergic (B) nervous system markers in the rat brain. (C, D): Western blot analysis of neurotrophic factors in the brain (C) and muscles (D). Abbreviations: AChE, acetylcholinesterase; ADMSC, adipose tissue-derived mesenchymal stem cells; AMMSC, amniotic membrane-derived mesenchymal stem cells; BDNF, brain-derived neurotrophic factor; ChAT, choline acetyltransferase; ChT1, choline transporter 1; DAT, dopamine transporter; D1R, dopamine 1 receptor; D2R, dopamine 2 receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDNF, glial cell-derived neurotrophic factor; IGF, insulin-like growth factor; m1AChR, muscarinic 1 acetylcholine receptor; nAChR α5, nicotinic acetylcholine receptor α5; nAChR β2, nicotinic acetylcholine receptor β2; NGF, nerve growth factor; TH, tyrosine hydroxylase; VAChT, vesicular acetylcholine transporter; VEGF, vascular endothelial growth factor; VMAT2, vesicular monoamine transporter 2.

Figure 5.

Transplanted stem cells increase microvessel density and muscle mass and enhance antioxidative capacity. (A, B): Density of microvessels (von Willebrand factor-positive) in the brain (A) and muscles (B), and gastrocnemius muscle mass (B). Scale bar = 50 μm. (C): Concentrations of TBARS. (D): Hepatic mCAT. ∗, significantly different from young rats (p < .05). #, significantly different from aged rats (p < .05). Abbreviations: ADMSC, adipose tissue-derived mesenchymal stem cells; AMMSC, amniotic membrane-derived mesenchymal stem cells; mCAT, mitochondrial catalase; TBARS, 2-thiobarbituric acid-reactive substances.

Figure 6.

Stem cell transplantation increases host stem cell proliferation, suppresses CCL11 expression, and restores neuronal integrity. (A): Double immunostaining for host nestin (stem cell marker) and Ki-67 (proliferating cell marker) in the brain of rats transplanted with ADMSCs. Scale bar = 25 μm. (B): Host stem cell counts. White bar, young rats; black bar, aged rats; striped bar, aged rats transplanted with AMMSCs; checked bar, aged rats transplanted with ADMSCs. ∗, significantly different from young rats (p < .05). #, significantly different from aged rats (p < .05). (C): Polymerase chain reaction and Western blot analyses of CCL11 and MAP2, respectively, in the brain. Abbreviations: ADMSC, adipose tissue-derived mesenchymal stem cells; AMMSC, amniotic membrane-derived mesenchymal stem cells; MAP2, microtubule-associated protein 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.