Endogenous Stem Cells in Homeostasis And Aging Part 1

Abstract In almost all human tissues and organs, adult stem cells or tissue stem cells are present in a unique location, the so-called stem cell niche or its equivalent, continuously replenishing functional differentiated cells. Those endogenous stem cells can be expanded for cell therapeutics using ex vivo cell culture or recalled for tissue repair in situ through cell trafficking and homing. In the aging process, inefficiency in the endogenous stem cell–mediated healing mechanism can emerge from a variety of impairments that accumulate in the processes of stem cell self-renewal, function, differentiation capacity, and trafficking through cell-autonomous intrinsic pathways (such as epigenetic alterations) or systemic extrinsic pathways. This review examines the homeostasis of endogenous stem cells, particularly bone marrow stem cells, and their dysregulation in disease and aging and discusses possible intervention strategies. Several systemic pro-aging and rejuvenating factors, recognized in heterochronic parabiosis or premature aging progeroid animal models, are reviewed as possible anti-aging pharmaceutical targets from the perspective of a healthy environment for endogenous stem cells. A  variety of epigenetic modifications and chromosome architectures are reviewed as an intrinsic cellular pathway for aging and senescence. A gradual increase in inflammatory burden during aging is also reviewed. Finally, the tissue repair and anti-aging effects of Substance-P, a peptide stimulating stem cell trafficking from the bone marrow and modifying the inflammatory response, are discussed as a future anti-aging target.

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Keywords Hematopoietic stem cell · Bone marrow stem cell · Endothelial precursor cell · Aging  Rejuvenation

1 Introduction

Aging is a natural phenomenon marked by a progressive decrease in a body’s homeostatic and regenerative functions, including age-related deterioration of stem cell functions [1]. Recent studies between young and old animals and humans have revealed age-related alterations in stem cell traits, stem cell pool size, and differentiation diversity [2–4]. In the aged, cell-autonomous epigenetic alterations in stem cells accumulate, which can cause senescence of stem/progenitor cells and reduce both the stem cell pool and stem cell function. Heterochronic parabiosis experiments between young and old animals have demonstrated the existence of age-related alterations in systemic and local environments, including systemic rejuvenating and pro-aging factors [5–7], that could control the function and lifespan of endogenous stem cells and stem cell niches. More prominently, experiments have revealed aged-related elevation of inflammatory cytokines and chronic inflammation [8–10], which could also affect stem cell function, stem cell niche, stem cell trafficking, and differentiation diversity. Those reports about aging and degenerative diseases such as Parkinson’s disease, Alzheimer’s disease, diabetes, chronic vascular disease, and osteoarthritis have pointed out possible links between stem cell status and disease etiology and between stem cell status and life expectancy.

In 2013, Carlos Lopez-Otin et al. [2] identified and categorized nine cellular and molecular hallmarks that contribute to the aging process: genomic instability,  telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Most age-related damage and deterioration accumulate gradually over time,  which has been extensively studied in the fields of cancer,  metabolic diseases, and degenerative diseases. In particular, the concept of stem cell exhaustion, through cell-autonomous intrinsic pathways or extrinsic pathways, could explain the integrative consequences of multiple types of aging-associated damage and the decline in regenerative potential [11, 12]. Progeroid mice and recent parabiosis experiments using young and old mice have demonstrated that systemic factors such as hepatocyte growth factor activator (HGFA) that are abundant in young mice can reverse the decline in neural and muscle stem cell function found in old or progeroid mice [13–15]. Those seminal works propose that systemic extrinsic factors, acting as a  pharmacological intervention, could stimulate stem cell rejuvenation and reverse the aging phenotype.

This review discusses endogenous stem cells in vivo,  especially bone marrow stem cells such as hematopoietic stem cells (HSCs), bone marrow stromal (or mesenchymal)  stem cells (BMSCs), and endothelial precursor cells (EPCs), based on their ontogeny, normal functions, and age-related functional alterations. Second, age-related alterations in systemic factors, candidates for anti-aging or pro-aging factors, are considered for their possible links to stem cell homeostasis and aging and for their potential as pharmacological targets to retard or control the aging process. Third, age-related alterations in the bone marrow stem cell niche are reviewed. Fourth, epigenetic changes to stem cells during aging, so-called cell autonomous stem cell aging, are reviewed and discussed in terms of histone modification, DNA methylation, and non-coding RNA. Fifth, the effects of enhanced inflammatory responses during aging are discussed, particularly in age-related degenerative diseases and defective tissue repair. Conclusions and perspectives for future research include a consideration of whether age-related deterioration in endogenous stem cells and their environment can be controlled and new pharmacological targets for human health.

2 Endogenous stem cells in the bone marrow and aging

Homeostasis, tissue repair, and regeneration are continuously maintained and stimulated by endogenous tissue stem cells in the body. Bone marrow is a stem cell reservoir for HSCs, BMSCs, EPCs, and other stem cells yet to be identified. The stem cells in the bone marrow are vitally involved in tissue regeneration, and senescence or loss of the function of those stem cells is the main cause of failure in tissue homeostasis and repair (Fig. 1) [4].

HSCs continuously supply blood cells, including immune cells, red blood cells, and white blood cells,  throughout life. Hematopoietic dysfunction emerges in age, resulting in decreased adaptive immune response and increased myeloid responses, and anemia. A prominent aging-associated alteration, so-called inflammaging, a  smoldering pro-inflammatory phenotype [9, 16], could also result from HSC dysfunction or loss of differentiation diversity. BMSCs are stromal cells in the bone marrow that might support hematopoiesis as stem cell niche cells as well as osteogenesis for bone formation. In the aged, BMSCs also decline, and their differentiation potential skews toward adipogenesis, resulting in yellow marrow and osteoporosis. EPCs, precursors to endothelial cells,  also decline during aging, causing impairment of vascular repair and impaired vascular niche function for HSCs.

2.1 HSC definition/ontogeny/aging

HSCs are the only cells that can produce the whole blood cell lineage. They generate * 1 9 1010 red blood cells and * 1 9 108 white blood cells every hour throughout life [11]. This continuous process of mature blood cell production, called hematopoiesis, involves the proliferation, self-renewal, and differentiation of HSCs and the egress of mature progenitor cells into the circulating blood [12]. Hematopoiesis occurs in ontogenetic flows. Primitive phases and definitive phases produce true HSCs. The emergence of primitive HSCs starts in the yolk sac at 30 days post-conception in humans and E7.5 in mice and then moves to the allantois and placenta. At 4 weeks post-conception (wpc) in humans and E10.5 in mice, HSCs lie in the aorta-gonad mesonephros, a region of the embryonic mesoderm that gives rise to definitive HSCs. HSCs are subsequently seeded into the placenta, thymus, and liver at 5 wpc in humans and E11 in mice, into the spleen at 8 wpc in humans and E14 in mice, and into the bone marrow at 12  wpc in humans and E18 in mice. After birth, HSCs exist only in the bone marrow and thymus, and the bone marrow supports the majority of hematopoiesis [17]. HSCs give rise to both common myeloid progenitor cells (CMPs) and common lymphoid progenitors (CLPs) to generate all blood cells. CMPs differentiate into monocytes, macrophages, dendritic cells, neutrophils, basophils, eosinophils,  erythrocytes, megakaryocytes, and platelets. CLPs differentiate into T cells, B cells, and NK cells (Fig. 2).

An HSC hierarchy can be defined based on bone marrow–repopulation capacity, differentiation potential, and multiple surface marker expression. In 1994, Spangrude et al. [18, 19] divided the population of HSCs into three multipotent types: long-term (LT)-HSCs, short-term (ST)- HSCs, and multipotent progenitors (MPPs). They identified those cells using a diverse cluster of differentiation markers, such as CD34, CD38, CD90, CD133, CD105, CD45,  and the stem cell factor c-kit. In 2005, to identify the functionally distinct subpopulation of HSCs from non-self-renewing MPPs, Morrison et al. [20, 21] reported that the signaling lymphocyte activation molecule (SLAM) family of receptors including CD150 (slamf1), CD48 (slamf2), CD229 (slamf3), and CD224 (slamf4) was differentially expressed among functionally distinct stem cells and progenitor cells in mice. In addition, weak staining with vital dyes such as Hoechst 33342 (side population) or rhodamine 123 can be used to isolate HSCs with small cell sizes. The ability of hematopoietic progenitors to proliferate and differentiate into colonies is examined with the colony forming unit (CFU) assay using methylcellulose-based semi-solid media in response to cytokine stimulation for lymphocytes (CFU-L), pre-B cells (CFU-Pre-B), granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM). To identify the cell cycle status, Ki-67  labeling, BrdU incorporation analysis, and the fluorescence ubiquitination-based cell cycle indicator (Fucci) system are used. Using the fusion of fluorescent proteins to the cell cycle–specific proteins geminin, cdt1, and p27, the Fucci system can visualize cells by staining cell nuclei in the G1, S/G2/M, and G0 phases of the cell cycle in cyan, green,  and red, respectively [22].

A variety of age-related cellular and molecular alterations in HSCs and hematopoiesis have been reported. In HSC function, aging leads to the over-proliferation of HSCs, whereas aging decreases stem cell numbers and cell cycle activity in skeletal muscle stem cells, germline stem cells, and neural stem cells [1, 23]. How those changes are controlled and regulated remains unclear. However, it was reported that aged HSCs showed decreased self-renewal,  loss of cell polarity, egress into the bloodstream, impaired homing ability, and myeloid- and platelet-biased differentiation [12, 24].

Age-related changes in systemic factors, intracellular signaling pathways, and cell cycle-controlling molecules also seem to be implicated in HSC self-renewal and differentiation diversity. TGF-b1 enhances myeloid differentiation rather than lymphoid differentiation [25], and the chromatin regulator Satb1, which is induced during lymphoid differentiation, decreases in aged HSCs [26]. Aged HSCs activate the non-canonical Wnt signaling pathway instead of the canonical Wnt pathway, which increases Wnt5a  expression and activates cell division control protein 42 (Cdc42) polarity. The regulation of Cdc42 polarity and distribution is important for regenerative capacity and bone marrow homing [27]. Also, in the aged, cell cycle checkpoints such as p16INK4a, BCL-2, BATF, and p53 are activated. Abolishing p16INK4a increases the regenerative potential of stem cells from bone marrow and the brain, and a  low level of p53 promotes stem cell maintenance, while a  high level induces cell death and senescence [28].

Reactive oxygen species (ROS) and nitric oxide (NO)  could also be important regulators in HSC aging. The accumulation of ROS in aged cells induces FOXO depletion, NF-jB activation, p38-mTOR activation, telomere shortening, DNA damage, and mitochondrial dysfunction [12]. Insulin and IGF activate the PI3K-Akt signaling pathway and phosphorylate FOXO, followed by inhibition of the expression of the anti-oxidant N-acetyl-L-cysteine [28]. After oxidative stress, HSCs increase NO levels,  which results in loss of self-renewal, abnormal proliferation, and malignancy [12, 29].

2.2 BMSCs in Young and old bone marrow

BMSCs, also generally called mesenchymal stem cells (BMSCs), do not express the HSC and EPC marker CD34,  but they reveal plastic adherent clonogenic properties and differentiate into a variety of cell types, such as osteoblasts,  chondrocytes, adipocytes, and myocytes, upon in vitro cell culture [30–32]. BMSCs express CD73, CD90, CD105, CD29, CD44, CD71, CD106, CD120a, CD124, CD56, and CD271 on their surfaces, but they lack CD11b, CD14, CD117, CD19, CD34, CD45, CD79a, and HLA-DR surface markers [33]. While in vivo BMSCs express CD146, SCA1, PDGFRa, CXCL12, and nestin, ex vivo aged BMSCs express CD106 and CD295 [34]. Enhanced expression of CD295, a leptin receptor, marks apoptotic cells and non–self-renewal cells.

BMSCs have self-renewal capacity, mobilize to injury sites, and participate in immune modulation, wound healing, and repair of almost all tissues [34–37]. The main physiological capacity of BMSCs is an immune modulatory function by releasing cytokines, which seems to be independent of traditional stem cell activity. In aging, the proliferation capacity, differentiation potential, and genomic stability of BMSCs decline. Whereas extensive HSC  aging research has been carried out, research into BMSCs in the aging process remains insufficient. In aged bone marrow, aberrations within the BMSC microenvironment,  such as chronic inflammation, result in fat deposits that coincide with a decrease in mesenchymal progenitors, bone loss, and fibrosis [38]. This age-dependent decline in BMSC function weakens its immune modulation capacity. BMSCs from the bone marrow of aged mice showed decreased colony-forming capacity. Moreover, BMSCs from aged bone marrow showed prominently reduced mobilization, possibly through downregulation of the phosphorylation of JNK signaling [39]. Therefore, maintaining a sufficient BMSC pool and competent BMSC  trafficking from the bone marrow is essential to a healthy marrow environment for inflammation modulation and also to facilitate tissue regeneration following several types of peripheral tissue damage. In the aged, decreased regeneration potential, enhanced autoimmune response, and enhanced inflammatory response could all be strongly interconnected with age-related BMSC decay and dysfunction [38].

2.3 EPCs in the bone Marrow and aging

EPCs in the bone marrow and circulating EPCs in peripheral blood can differentiate into endothelial cells and form the endothelial lining of the vasculature. Both EPCs and HSCs in the bone marrow are derived from hemangioblast [40]. EPCs in the bone marrow express CD34, CD133, and VEGFR2 (KDR/Flk1). After moving from the bone marrow to the blood, circulating EPCs lose their progenitor capacity and start endothelial differentiation,  expressing von Willebrand factor, CD31, CD144, VEcadherin, and eNOS [41, 42].

EPCs can be mobilized from the bone marrow and play a pivotal role in tissue repair with mechanisms to regenerate and maintain the endothelium by regulating coagulation, arterial tone, permeability, vessel growth, and inflammation. As aging progresses, the number of EPCs and their function decreases with increased oxidative stress,  inflammation, senescent phenotype oxidized low-density lipoprotein (ox-LDL), and telomere shortening, which eventually increases the risk for vascular diseases such as atherosclerosis and cardiovascular disease [43]. EPCs from aged humans are sensitive to oxidative stress, probably due to reduced levels and activity of the antioxidant enzyme glutathione peroxidase-1 (GPX1), which then lowers cell survival [44]. Ox-LDL, a risk factor for cardiovascular disease, accumulates with age and reduces the survival and function of EPCs by inhibiting eNOS expression and activity [45]. Aged EPCs are identified by their cell survival ability and the colony-forming unit assay, even though mechanisms of EPC aging have not been sufficiently studied. Therefore, EPC dysfunction with aging could also be interconnected with delayed repair of ischemic vascular damage and a higher risk of cardiovascular disease.

3 Systemic rejuvenation factors and pro-aging factors

Systemic change produces differences between young and old people and between healthy and diseased individuals. Active factors in stem cells can systemically and directly influence their fitness and guide their destinies.

Heterochronic parabiosis experiments have elucidated the presence of systemic rejuvenating and pro-aging factors and their alterations with age (Fig. 3). In 1864, the French physiologist Paul Bert carried out heterochronic parabiosis experiments on albino rats to study the effects of aging. Parabiosis (from the Greek words, para ‘‘besides’’ and bios ‘‘life’’) uses a surgical technique to physically connect the blood vessels and create a shared circulating system for two living organisms of different ages [5]. In the 1950s,  researchers found that old mice experienced rejuvenating effects, and the young mice had shorter than average lifespans in between surgically connecting two animals. In the 1970s, parabiosis experiments were banned by animal research regulations. However, they became active again in the 2000s for research on aging. Recent parabiosis studies have shown that rejuvenation factors in the blood can turn back the stem cell clock: the functions of old stem cells were rejuvenated, and younger stem cell functions were weakened when they were exposed to young or old serum,  respectively. Systemic rejuvenating and pro-aging factors are summarized in Table 1. Growth differentiation factor (GDF) 11, oxytocin, bursicon, and HGFA are all candidate systemic rejuvenating factors and C–C motif chemokine (CCL) 11 and b2-microglobulin (B2M) are pro-aging factors.

3.1 GDF11

The rejuvenating effect of GDF11 was first identified in parabiosis experiments. Bone morphogenetic protein 11,  another name for GDF11, was reported as a circulating factor that reversed age-related cardiac hypertrophy in mice [6]. GDF11, a TGF-b superfamily member, decreases during the aging process. Restoring youthful GDF11

However, in 2016–2017, conflicting reviews were published suggesting that GDF11 induced wasting of skeletal and cardiac muscle [48–51] and that GDF11 does not decline in rats or humans during aging [48]. Aging is a lifelong, complex phenomenon that accumulates over time. Therefore, more intensive cause-and-effect studies should be conducted to decisively elaborate the role of GDF11 as a  rejuvenating factor.

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