Aging puzzle: A closer look on the complex dilemma of autologous stem cell therapy

Abstract

Regenerative medicine is a promising therapeutic avenue for previously incurable diseases. As the risk of chronic and degenerative diseases significantly increases with age, the elderly population represents a major cohort for stem cell-based therapies. However, the regenerative potential of stem cells significantly decreases with advanced age and deteriorating health status of the donor. Therefore, the efficacy of autologous stem cell therapy is significantly compromised in older patients. To overcome these limitations, alternative strategies have been used to restore the age- and disease-depleted function of stem cells. These methods aim to restore the therapeutic efficacy of aged stem cells for autologous use. This article explores the effect of donor age and health status on the regenerative potential of stem cells. It further highlights the limitations of stem cell-based therapy for autologous treatment in the elderly. A comprehensive insight into the potential strategies to address the “age” and “disease” compromised regenerative potential of autologous stem cells is also presented. The information provided here serves as a valuable resource for physicians and patients for optimization of stem cell-based autologous therapy for aged patients.

Key Words: Regenerative medicine; Autologous stem cell therapy; Regenerative potential; Aging; Donor age; Optimizing therapy; Aged patients

Core Tip: Stem cell-based therapies, particularly autologous stem cell therapy, hold strong promise for treating age-related degenerative diseases by reducing the risk of immune rejection and graft-vs-host disease. However, the therapeutic efficacy of patient-derived stem cells is often compromised by aging and disease-related decline in their regenerative capacity. To address these limitations, several strategies have been developed, including hypoxic preconditioning, genetic modification, growth factor supplementation, three-dimensional culturing, hybrid approaches, use of bioactive compounds, mesenchymal stem cell-derived exosomes, targeted activation of signaling pathways and cryopreservation of youthful stem cells. These approaches aim to restore or preserve stem cell functionality, enhancing therapeutic outcomes in elderly patients.

INTRODUCTION

Promising advances have been made in the dynamic field of regenerative medicine, offering new avenues for the treatment of various diseases and disorders. Stem cell-based therapy has emerged as an alternative, particularly for conditions that cannot be treated with conventional medicines. Among the diverse array of adult stem cells, mesenchymal stem cells (MSCs) have gained significant attention due to their remarkable results in pre-clinical and clinical studies. MSCs were originally isolated from bone marrow; however, further studies have identified additional sources, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord blood (UCB), cord tissue, and Wharton’s jelly. This expanded repertoire of MSC sources enhances their accessibility and applicability for various therapeutic interventions. MSCs exhibit high proliferative potential and possess remarkable capacity for differentiation into the mesodermal lineage (such as osteocytes, adipocytes and chondrocytes) as well as ectodermal (neurons) and endodermal (hepatocytes) lineages. In addition, MSCs possess immunomodulatory and immunosuppressive capabilities and actively secrete myriad growth factors and cytokines, contributing to their role as efficient tools in regenerative medicine. These features position MSCs as versatile, efficient and promising treatment options for various medical applications within the field of regenerative medicine.

As people age, they become more susceptible to degenerative diseases such as neurological (e.g., Alzheimer’s disease), cardiovascular (e.g., atherosclerosis), musculoskeletal (e.g., osteoarthritis), and metabolic (e.g., diabetes) disorders. The increasing prevalence of age-related ailments poses substantial health challenges for the aging population. This observation aligns with the idea that the aging population may be particularly interested in therapies that have the potential to address age-related conditions and improve overall health. The pre-clinical and clinical studies highlight the potential of adult stem cells (e.g., MSCs) in treating age-related diseases. Therefore, a significant segment of the population seeking stem cell-based therapies likely comprises elderly individuals.

Autologous stem cell therapy involves the use of a patient’s own stem cells for medical use. Therapeutically, autologous stem cell-based therapy is preferred because it offers lower risk of rejection, no ethical concerns, reduced risk of infection and avoidance of graft-vs-host disease. While autologous stem cell therapy offers these advantages, there are also challenges. For example, the harvesting and processing of autologous stem cells can be a time-consuming and complex procedure. Most importantly, the potential of patient’s own stem cells may be compromised due to donor “age” and “disease status”. The natural process of physiological aging may exert detrimental effects on both the quantity and quality (i.e., regenerative capabilities) of stem cells[1]. With increasing donor age, the number of stem cells declines, their ability to regenerate and repair tissues deteriorates, and cells undergo apoptosis (programmed cell death) and senescence (cellular aging). The overall regenerative potential of aged stem cells ultimately declines due to these inherent phenomena. The reduced potential of aged stem cells poses significant challenges in harnessing the full therapeutic potential of stem cells for addressing age-related degenerative diseases. Similarly, the health status of the donor negatively influences the regenerative potential of stem cells[2]. This means that autologous stem cells harvested from elderly individuals may not be the most effective option for maximizing the benefits of stem cell therapy.

Recently, hypoxia, caloric restriction, heat shock, cryopreservation and different preconditioning strategies have been used to restore the regenerative potential of aged stem cells[3-5], thereby enhancing the therapeutic potential of aged stem cells. Additionally, genome editing of stem cells install specific characteristics that can optimize their functionality for desired therapeutic outcomes.

The current review is focused on examining the effect of donor “age” and “health status” on the regenerative potential of stem cells. The review aims to explore the limitations associated with utilizing autologous stem cells in elderly patients. It will also discuss different methods that can be used to improve the “age” and “disease” compromised regenerative potential of aged stem cells. In addition, cryopreservation will be discussed as an anti-aging strategy. The review aspires to serve as a valuable resource for physicians and patients for the optimization of autologous stem cell therapies tailored specifically for aged patients.

STEM CELL-BASED THERAPIES: AN UPDATE

Cell therapy is a medical procedure in which autologous or allogenic cellular material is transferred into a patient for medical purposes. This treatment approach aims to combat diseases and improves patient health by utilizing cells. Cell-based therapies have revolutionized the field of regenerative medicine and are currently widely utilized as effective therapeutic modalities. Cell-based therapies encompasses the use of stem cells and non-stem cells[6]. Non-stem cell-based therapies typically use somatic cells sourced from the body such as chondrocytes, fibroblasts, keratinocytes, pancreatic islet cells, hepatocytes, and immune cells, such as macrophages, natural killer cells, dendritic cells, and T cells. It is important to note that non-stem cell-based therapies rely on cells that have already undergone specialization, which limits their ability to proliferate and further differentiate. Non-stem cell-based therapies are typically focused on replacing or repairing cells that are similar to those from which they are derived. For instance, immune cells may be used to boost the body’s immune response, chondrocytes for repairing cartilage, or fibroblasts for regenerating skin tissue. Additionally, these specialized cells may indeed have a reduced life expectancy and could be more susceptible to aging and injury compared to stem cells. This inherent limitation underscores the need for ongoing research and development efforts to enhance the efficacy and longevity of non-stem cell-based therapies, as well as to explore alternative approaches that may mitigate these challenges[7]. Despite their restricted proliferative and differentiation capacities, therapies based on non-stem cells play crucial roles in regenerative medicine by replacing or repairing specific cell types, such as immunological, cartilage, or skin cells. These therapies contribute significantly to address a wide range of medical conditions and to improve patient outcomes.

Stem cells are unique in that they are unspecialized cells with the remarkable capacity to differentiate into various cell types. This differentiation process allows stem cells to give rise to specialized cells such as muscle cells, blood cells, neurons, and many others. This versatility makes stem cells invaluable in regenerative medicine, as they hold the potential to repair or replace damaged tissues and organs in the body. The high self-renewal ability of stem cells allows them to continuously divide and produce more stem cells of the same type. Therefore, stem cell-based therapies present a greater potential for regenerative medicine and a wider range of applications than non-stem cell-based therapies. It offers new hope to people with incurable diseases, where current therapeutic approaches focus on controlling the disease rather than treating it[8]. Autologous stem cells, which originate from the same patient receiving treatment, are used in most studies. The deployment of autologous stem cells has the benefit of avoiding ethical dilemmas and immunological rejection compared to allogenic stem cells[9]. Absolutely, stem cells possess the remarkable ability of infinite self-renewal, allowing them to continuously produce more stem cells of the same type. This characteristic distinguishes them from non-stem cells and greatly enhances their potential for regenerative medicine applications. Stem cell-based therapies offer a broader range of possibilities compared to non-stem cell-based therapies due to their capacity for differentiation and self-renewal. Furthermore, the use of autologous stem cells, which are derived from the same individual receiving treatment, is common in many studies. This approach offers several advantages, including the avoidance of ethical concerns associated with the use of embryonic stem cells and a lower risk of immune rejection compared to allogenic stem cells sourced from a different donor. As a result, autologous stem cell therapy holds significant promise, offering new hope to individuals with incurable diseases by providing potential treatments where conventional approaches may focus only on disease management rather than cure.

The ultimate goal of autologous stem cell-based therapy is to improve the capacity of the body to heal itself by stimulating, regulating, and altering the population of endogenous stem cells and replenishing the cell pool towards tissue regeneration and homeostasis. Since the introduction of the concept of stem cells, which possess special abilities to differentiate and self-renew, they have been extensively studied as a possible source for regenerative medicine to treat a wide range of diseases and injuries[9]. German zoologists Valentin Haecker and Theodor Heinrich Boveri first coined the term stem cell in 1868. Their goal was to find the unique cell population in the embryo that had the ability to differentiate into more specialized cells. This marked the beginning of the journey of stem cell therapy. Stem cell therapy technology has advanced over the years to treat a wide range of incurable diseases as a new therapeutic agent of regenerative medicine, such as pulmonary dysfunctions, neurological disorders, reproductive disorders, metabolic diseases, cardiovascular conditions, skin burns, and age-related disorders[8,10]. The historical milestones of stem cell-based treatments are highlighted in Table 1[8,10-12].

Table 1 Historical milestones of stem cell-based therapies[8,10-12].

Year	Discovery
1868	The earliest known use is by Ernst Haeckel (1868), referring to unicellular organisms as the origin of multicellularity[10]
1902	First description of hematopoietic progenitor cells by Franz Ernst Christian Neumann and Alexander A Maximov[12]
1939	First report of bone marrow transplantation for aplasmic anemia; first attempt failed
1957	Dr. E Donnall Thomas performed the first allogeneic hematopoietic stem cell transplantation
1958	First transplantation of stem cell for the treatment of radioactive exposure by George Mathe
1960s-1970s	Foundational works on stromal stem cells led by Friendenstein’s team
1972	The successful allogeneic transplants (first successful cases) for aplastic anemia
1981	The first murine ESCs were established by Evans and Kaufman and GR Martin
1991	Stromal stem cells were renamed as “Mesenchymal stem cells” by AI Caplan
1998	James Thomson isolated the first ESCs from human
2007	Discovery of iPSCs by Shinya Yamanaka and Kazutoshi Takahashi
2010	First clinical trial using ESC-derived OPC1 for the treatment of spinal cord injury conducted by Geron (NCT01217008)
2015	First case report of cardiac progenitors produced from human ESCs for severe heart disease
2017	First report of using iPSC-derived retinal cells in treatment of macular degeneration
2022	Reported 10 years follow up data on OPC1 administration for the treatment of spinal cord injury

ESC: Embryonic stem cell; iPSCs: Induced pluripotent stem cells.

Stem cell therapy is a complex biological process, as it requires scientists to identify a viable, safe, and easily accessible source of stem cells capable of proliferation and differentiation into the desired lineage. Therefore, selecting the ideal type of stem cells for a specific treatment requires careful consideration[12,13]. MSCs, a type of multipotent stem cells, exist in adult tissues such as umbilical cord tissue, bone marrow, dental pulp, UCB and adipose tissue. MSCs have been reported to express some ectodermal/endodermal markers under specific experimental conditions, although functional engraftment as neurons or hepatocytes remains controversial. MSCs are preferred for treating age-related disorders due to their unique biological properties and effective therapeutic potential (Table 2). They have attracted a widespread interest due to ease of isolation, proliferation, and differential potential[14]. Beside these characteristics, MSCs also exhibit anti-inflammatory, immunomodulatory, anti-apoptotic, and angiogenic characteristics. Therefore, they are used to treat a wide range of autoimmune, degenerative, and inflammatory disorders[14]. Moreover, they possess the ability to regulate the immune system and reduce inflammation, which are essential factors in aging and age-related disorders. Enhancing the regeneration capability and overall health of aged tissues is one of the possible advantages of MSC-based therapy, which may help solve the aging puzzle[14,15].

Table 2 Characteristics of mesenchymal stem cells supporting their use for age-related disorders.

Feature	Benefit for age-related disorders
Multilineage differentiation	Regenerates multiple tissue types damaged by aging
Low immunogenicity	Reduces risk of immune rejection
Immunomodulation	Controls chronic inflammation common in aging
Ease of isolation and expansion	Facilitates clinical application
Exosome secretion	Delivers anti-inflammatory, antioxidant and regenerative factors
Combats oxidative stress	Protects cells from age-related damage
Rejuvenation potential	Restores function of aged stem cells

The potential of MSC based cellular therapies has been tested in many clinical trials since 1995. As of September 6, 2025, 1609 trials had been registered using MSCs on ClinicalTrials.gov (https://clinicaltrials.gov/). These registered clinical trials used MSCs to treat a range of diseases, including coronavirus disease 2019, osteoarthritis, graft-vs-host disease, spinal cord injury, stroke, myocardial infarction, and age-related diseases. Of all the clinical trials that were registered, 52 were listed as early phase I, 887 as phase I, 795 as phase II, 106 as phase III, 14 as phase IV, and 174 as not applicable. Figure 1 depicts the status of different clinical trials using MSCs, although the data are incomplete due to lack of information regarding the status of several of these clinical studies.

Figure 1 Clinical trials status of mesenchymal stem cell-based therapies. This pie chart displays the distribution of clinical trials related to mesenchymal stem cells, showing various statuses of these trials. The majority of trials are either completed (546 trials) or active but not recruiting (428 trials). A smaller number of trials are enrolled by invitation (281 trials) and seeking participants (62 trials). There are also trials that are suspended (24 trials), terminated (72 trials), withdrawn (11 trials), or have an unknown status (36 trials). Each section of the chart is color-coded to indicate the respective trial status. MSCs: Mesenchymal stem cells.

Outcomes of clinical trials are inconsistent due to lack of standardization. These studies have used different MSC sources, number, route of transplantation, time of reporting, frequency of transplantation, as well as the patient’s condition, disease stage, and immunological status[13]. Several challenges confront MSC-based therapies in clinical settings, including different criteria for MSC identity and quality, heterogeneity, and variability in MSC, poor in vivo survival, and engraftment rates, ethical and legal dilemmas, and possible tumorigenicity and immunogenicity risks. Therefore, strategies are required to overcome these challenges to ensure optimized MSC therapy. These strategies aim to standardize the protocols for MSC isolation, characterization, maintenance, dosage, transplantation and most importantly, outcome assessment methods. MSCs have the potential to provide innovative and efficient treatment for a range of human diseases, but before it can be extensively used in clinical practice, it must first undergo careful evaluation and optimization[13,16].

AGED PATIENTS: PRIMARY TARGET POPULATION FOR STEM CELL-BASED THERAPIES

Aging is a multifactorial and intricate process, characterized by the time-dependent deterioration of physiological functions, which increases susceptibility to diseases and ultimately leads to death. Aging is a hallmark of telomere shortening, loss of proteostasis, cellular senescence, mitochondrial dysfunction, genomic instability, stem cell depletion, impaired intercellular communication, dysregulated nutrient sensing, and epigenetic changes[17]. One of the most affected aspects of aging is the regenerative potential of stem cells, which plays a central role in tissue repair and homeostasis. Adult stem cells maintain and repair tissues throughout the life of an organism. They are found in special microenvironments within tissues called stem cell niches. The proliferative ability (self-renewal) and differentiation potential of these cells into distinct and specialized cell types is important for repair, regeneration, and maintenance of tissue and organ function. However, as we age, these cells lose their inherent ability for regeneration, which compromises tissue repair and the maintenance of homeostasis. Aged stem cells may not have the same strong ability to regenerate, repair and sustain the host system against the damaging effects of aging[18]. Normally, stem cells respond to their niche and growth signals in a manner that is sufficient to maintain tissue and organ integrity. However, aging progressively produces a bias in stem cell function by slowing response to signals that results in delayed repair of organs and tissues. Overall, with organismal aging, stem cells also become aged, and their self-renewal and differentiation potential decline, resulting in impaired regeneration and homeostasis of organs in aged individuals. At the molecular level, these changes are due to intrinsic damages, aberrant DNA alterations, and changes in the tissue microenvironment, both the niche and circulating factors[19].

The depletion of stem cell activity plays a major role in the emergence of many age-related disorders, including arthritis, osteoporosis, neurodegenerative disease, cardiovascular disease, cancer, and diabetes. These diseases often progress slowly but severely diminish the quality of life of elderly people and increase healthcare costs. The average human life expectancy has increased significantly because of improvements in general lifestyle, medical research, health care practices, hygiene and food availability. However, previous research focused heavily on the extension of life rather than quality of life. It resulted in extension of life without delaying the aging process and without significantly reducing age-related disease. While human life expectancy has increased dramatically, the quality of life has not improved proportionally, resulting in a higher risk of diseases, disabilities, dementia, and other degenerative conditions before death. Most of the chronic and degenerative diseases of aging cannot be effectively cured with conventional medicines, such as pharmaceutical and other standard medical therapies. The drastic increase in elderly medical patients has resulted in the development of alternative treatment options that can revive the health and functionality of aged tissues and organs. Stem cell-based therapies have emerged as an alternative treatment option for the elderly[20].

Overall, the aging puzzle highlights an inconsistency in regenerative medicine: As organisms grow older, the risk of chronic and degenerative diseases steadily increases, while the regenerative capacity of stem cells declines. This makes elderly individuals the primary candidates for stem cell-based therapies, yet the very decline in stem cell function with age limits the effectiveness of autologous treatments. In other words, the people who need stem cell therapies most often have the cells least capable of supporting repair and regeneration, creating a major therapeutic challenge in addressing age-related disorders. Figure 2 shows the schematic illustration of the aging puzzle in autologous stem cell therapy.

Figure 2 The aging puzzle in autologous stem cell therapy. Schematic representation illustrating the complex interplay between aging, health status, and the regenerative potential of autologous stem cells. As the donor’s age increases and health status decreases, the regenerative capabilities of stem cells are compromised. This figure emphasizes the challenges associated with utilizing autologous stem cell therapy in the elderly, prompting the exploration of alternative strategies to enhance regenerative potential.

Stem cell-based therapies hold great promise for restoring tissue function in aging individuals. For example, osteoarthritis, a common condition in older adults, leads to cartilage breakdown and reduced mobility. Stem cell therapy can help regenerate cartilage tissue and improve joint function in such patients. Likewise, aging is a major risk factor for neurological disorders such as Alzheimer’s disease, which impairs memory and cognition. Transplanting neural stem cells offers the potential to repair damaged brain regions and improve cognitive abilities[18]. Because aging itself contributes to tissue degeneration, elderly patients represent the main target group for stem cell-based therapies, as they can benefit from tissue rejuvenation and from the prevention or treatment of age-related diseases. The main goal of stem cell therapy is to restore and rejuvenate tissues; however, delivering functional stem cells to elderly patients is challenging because the regenerative potential of stem cells is strongly affected by external factors, many of which lose effectiveness with age[18,21]. Figure 3 highlights the limitations associated with using autologous (self-derived) stem cells in older individuals. Recognizing these limitations is an essential step toward designing more effective strategies to combat age-related degenerative conditions.

Figure 3 Limitations of autologous stem cell therapy in the elderly. This figure outlines the key limitations of autologous stem cell therapy in elderly patients. These include reduced stem cell numbers, compromised functionality due to aging, immune and frailty-related changes, challenges in harvesting quality cells, and varied therapeutic responses. The number of stem cells decreases with age, limiting therapy options. Aged stem cells also have lower regenerative potential and changed gene expression, which decreases their therapeutic potential. Age-related frailty and immune system alterations can also impact therapy. Harvesting stem cells from elderly people is difficult and risky, which may lower cell quality. Therapy outcomes vary for older people, and physical health is a major component.

REGENERATIVE POTENTIAL OF STEM CELLS VARIES WITH DONOR AGE

Donor age strongly influences stem cell potential, which directly affects the outcomes of stem cell-based therapies in elderly patients. Stem cells from older individuals exhibit reduced self-renewal, differentiation, and proliferative capacity, along with increased apoptosis and senescence. Age is therefore associated with a diminished capacity for reparative and regenerative processes. As individuals grow older, their ability to maintain homeostasis and tissue turnover declines. It is thought that the decreased functional capacity of tissue-resident stem cells is responsible for poor organ and tissue healing at the organismal level. Due to age-related alterations, stem cells from an older donor are less effective in promoting tissue rejuvenation compared to those from younger donors. Specifically, aged stem cells have significantly reduced self-renewal and differentiation potential, rendering them less responsive to niche-derived cues and growth factor signaling. Studies in both animal and human tissues consistently demonstrate that donor age decreases the yield of isolated stem cells, the number of colonies formed, and their proliferation and differentiation potential. Additionally, stem cells from aged donors show pronounced features of cellular aging, such as increased apoptosis, decreased superoxide dismutase activity, shorter telomeres, elevated reactive oxygen species (ROS) levels, and impaired functional abilities[2]. Telomere shortening is one of the most significant indicators of aging. Telomeres progressively shorten because of the incomplete replication of linear chromosomes during each round of DNA synthesis and cell division. Once the telomeres reach a critically short length, cells lose their ability to proliferate and enter senescence. In stem cells, telomere length decreases from an average of 10 kbp to approximately 7.1 kbp from early to late passage cells, with an estimated progressive loss over time. In MSCs, telomere shortening is reported to occur at a rate of approximately 100 bp every two passages. This progressive telomere shortening is one of the primary causes of aging, as short telomeres triggers chromosomal senescence and reduced cell viability (Figure 4). Collectively, these modifications compromise stem cell safety and efficacy from elderly donors for treating age-related disorders[18].

Figure 4 The cellular consequences of telomere shortening through three distinct pathways: Telomere inhibition, sub-telomere amplification, and telomere recombination. In telomere inhibition, progressive shortening triggers DNA damage responses and genomic instability, leading to chronological and replicative aging. Sub-telomere amplification may transiently lengthen telomeres but induces replication stress and genomic instability, ultimately resulting in renewed telomere shortening and replicative senescence. Telomere recombination also promotes telomere lengthening; however, it causes DNA replication errors and genomic instability, leading to non-telomeric damage, replicative senescence, and aging.

Several studies have evaluated the negative effect of donor age on regenerative potential of stem cells. For example, Strässler et al[22] observed that donor age influences the markers of aging, reprogramming efficiency, and the differentiation potential of induced pluripotent stem cell (iPSC)-derived cells. Their work further highlighted that donor age is a critical factor that varies depending on the stem cell source. They emphasized the need for more research on the mechanisms and implications of cardiovascular regenerative cell aging. Similarly, Carvalho et al[23] reported that human UCB cells derived from young donors exhibited lower senescence, higher proliferation and greater osteogenic support compared to cells derived from old donors. Based on these findings, they recommended that young donors are better suited for stem cell-based applications. Choudhery et al[1] also demonstrated that donor age negatively affects the regenerative potential of stem cells. This study showed that adipose tissue-derived MSCs from elderly donors displayed higher senescence, reduced viability and proliferation, lower antioxidant enzyme activity, and impaired differentiation capacity compared to those from younger donors[1]. Consistent with these findings, another study demonstrated that bone marrow-derived MSCs from aged mice (23-24 months old) exhibited significantly reduced wound healing capacity, impaired angiogenesis, diminished proliferation, and weakened anti-apoptotic functions[24]. Major findings of other studies are summarized in Table 3[1,23,25-36]. While most studies report a decline in stem cell function with increasing donor age, some studies show no significant age-related effects. These conflicting results may be attributed to variations in tissue sources, isolation protocols, and experimental conditions, such as in vitro vs in vivo models. Differences in study design, sample size, and methodology could also contribute to these discrepancies.

Table 3 Studies on how donor age affects the regeneration potential of stem cells.

	Title of study	Type of cells used	Sample size	Study type	Major findings	Ref.
1	Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential	ADSCs	122	In vitro	Donor age had little effect on self-renewal capacity, proliferation, cell yield, and osteogenic capacity of ADSCs. ADSCs are a reliable resource for gene therapy applications involving both autologous and allogeneic cells	[25]
2	Impact of donor age on the osteogenic supportive capacity of mesenchymal stromal cell-derived extracellular matrix	ADSCs	4	In vitro	Older donors showed reduced osteogenic support	[23]
3	Transplanting cells from old but not young donors causes physical dysfunction in older recipients	ADSCs	12	In vivo	Old ADSCs impaired physical function in recipients	[26]
4	Age affects the paracrine activity and differentiation potential of human adipose-derived stem cells	ADSCs	8	In vitro	Older ADSCs had reduced paracrine function and differentiation	[27]
5	Effect of donor age and 3D-cultivation on osteogenic differentiation capacity of adipose-derived mesenchymal stem cells	ADSCs	11	In vitro	Younger ADSCs showed stronger osteogenesis; older cells declined	[28]
6	Age-related changes in the regenerative potential of adipose-derived stem cells isolated from the prominent fat pads in human lower eyelids	OADSCs	20	In vitro	Regenerative potential declines with donor age	[29]
7	Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells	ADSCs	18	In vitro	No significant correlation with age	[30]
8	Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation	ADSCs	40	In vitro	Minimal effect on chondrogenic/osteogenic potential	[1]
9	The effect of age on the regenerative potential of human eyelid adipose-derived stem cells	OADSCs	13	In vitro	Aging reduced osteogenic, chondrogenic, and wound-healing abilities	[31]
10	The influence of aging on the regenerative potential of human adipose derived mesenchymal stem cells	ADSCs	28	In vitro	Older ADSCs had reduced proliferation and differentiation	[32]
11	Adipose-derived mesenchymal stem cells from the elderly exhibit decreased migration and differentiation abilities with senescent properties	Subcutaneous ADSCs	24	In vitro	Elderly ADSCs had reduced migration and higher senescence	[33]
12	Human periosteal derived stem cell potential: The impact of age	Periosteal stem cells	8	In vitro	Aging altered markers and bone remodeling gene	[34]
13	Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells	BM-MSCs	53	In vitro	No correlation with donor age	[35]
14	Aging alters tissue resident mesenchymal stem cell properties	ADSCs	40	In vitro	Aging reduced differentiation ability	[36]

ADSCs: Adipose-derived stromal cells; 3D: Three-dimensional; OADSCs: Orbital adipose-derived stromal cells; BM-MSCs: Bone marrow-derived mesenchymal stem cells.

EFFECT OF DONOR HEALTH STATUS ON STEM CELLS

Donor health is as important as donor age in determining stem cell quality. A healthy donor is essential for both autologous and allogeneic therapies. Allogeneic therapies employ donor cells to treat a variety of diseases, whereas autologous therapies use a patient’s own cells. In both cases, high-quality donor material is critical for reliable process development and therapeutic outcomes. Healthy donor cells provide several advantages over diseased cells: They are more accessible, biologically relevant, and functionally intact. They also offer more appropriate functional profiles for assay development and quality evaluations[37]. In healthy individuals, the stem cell niche is tightly regulated; however, disease conditions such as cardiovascular disease, diabetes, glucocorticoid imbalance, osteoporosis, arthritis, and cancer can alter stem cell number, proliferation, migration, adhesion, and angiogenesis[2,38]. Similarly, stem cells isolated from the adipose tissue of obese individuals demonstrate poor yield, reduced motility, and impaired angiogenesis. Age and disease often act synergistically to compromise regenerative potential. Aging and illness are associated with increased ROS production, telomere shortening, decreased telomerase activity, and elevated expression of apoptotic and senescent markers. These effects are more pronounced in aged individuals, whose stem cells are less capable of repair and regeneration compared to those of younger donors[2].

The donor’s genetic background is another important determinant of stem cell functionality. Genetic polymorphisms, such as single nucleotide polymorphisms (SNPs), contribute to functional heterogeneity and can affect gene expression, immunogenicity, differentiation potential, and drug responsiveness. Although the effects of many individual SNPs remain unclear, they are predicted to negatively influence stem cell-based therapies depending on the stem cell type and intended application. SNPs may alter the immunomodulatory properties of MSCs, affecting their ability to prevent inflammation and avoid rejection. For instance, SNPs within the p16INK4a-pRB pathway are known to regulate the cell cycle and senescence. In MSCs, polymorphisms in this pathway could lead to an increased susceptibility to senescence, reducing their regenerative potential. Similarly, SNPs in genes involved in oxidative stress response, such as superoxide dismutase or glutathione peroxidase, can influence the stem cells’ ability to withstand oxidative damage commonly associated with aging. They may also influence gene expression during MSC differentiation, migration, and homing. Gene-editing approaches offer a potential solution, enabling modification of stem cells to overcome these genetic barriers and tailoring them for personalized therapies[39]. Even in autologous applications, healthy donor material is often used in research and manufacturing to establish reliable production processes. However, the demand for dependable, high-quality donor material significantly exceeds the supply of patient donations. This growing need is driven by the rapid development of new drugs and clinical pathways. Therefore, identifying alternative sources and innovative techniques for acquiring healthy donor material remains a critical challenge for advancing cell-based therapies.

EMERGING STRATEGIES TO AUGMENT REGENERATIVE POTENTIAL OF STEM CELLS IN ELDERLY PATIENTS

Because donor age and health negatively affect the regenerative capacity of stem cells, scientists have proposed various methods to effectively rejuvenate elderly stem cells, either from autologous or allogenic donors for cell-based therapies. These strategies include hypoxic conditioning, genetic modifications (GMs), growth factor supplementation, hybrid stem cell therapy, use of bioactive compounds, MSC-derived exosomes, activation of signaling pathways, three-dimensional (3D) culturing, and cryopreservation[18,19]. Collectively, these approaches aim to restore stem cell function and enhance their reparative and regenerative capacity (Figure 5). However, these approaches also introduce challenges, such as addressing the heterogeneity of MSC populations, ensuring the safety and efficacy of cell products, and minimizing risks in elderly patients. Table 4 compares and summarizes each strategy in term of their relative efficacy, practicality, cost-effectiveness, potential applications, and major bottlenecks.

Figure 5 Overview of emerging approaches to restore the regenerative capacity of stem cells compromised by age and health status. These include hypoxic conditioning, genetic modifications, activation of signaling pathways, growth factor supplementation, bioactive compounds, exosome therapy, hybrid cell strategies, and advanced three-dimensional culture systems. Applying these methods to aged stem cells may rejuvenate their function and open new avenues for therapeutic interventions in elderly patients. 3D: Three-dimensional; MSCs: Mesenchymal stem cells.

Table 4 Comparison of emerging strategies to augment regenerative potential of stem cells in the elderly.

Strategy	Efficacy	Practicality	Cost-effectiveness	Major bottlenecks
HPC	Effective for rejuvenating stem cells by mimicking natural hypoxic environments, improving mitochondrial efficiency and reducing ROS accumulation	Relatively simple to implement in vitro but translating to in vivo conditions is challenging due to varying oxygen tensions	Low cost for in vitro applications; however, in vivo translation may incur higher costs	Difficulty in replicating in vivo oxygen conditions; risk of increasing genomic instability or harmful factor secretion in aged cells
Genetic modification (CRISPR/Cas9)	Highly effective in precisely editing genes associated with aging and cellular senescence, improving stem cell function	Technically challenging; requires specialized expertise and equipment	High initial cost for setup, but cost-effective for large-scale genetic modifications	Off-target effects; potential risk of inducing tumorigenesis or mutations in aged stem cells
Growth factor supplementation	Effective for enhancing cell survival, proliferation, and differentiation, especially in aged cells	Easy to apply, but requires careful management of dosages and delivery systems	Moderate to high cost depending on the growth factor and delivery system used	Short half-life of growth factors limits their long-term effectiveness; managing consistent delivery in vivo is challenging
Bioactive compounds	Promising for enhancing stem cell function through modulation of pathways like oxidative stress and mitochondrial function	Non-invasive and easy to implement, but requires high doses for efficacy	Relatively low cost for sourcing and application, though clinical use may require further investment	High doses required for efficacy, potentially leading to toxicity in elderly patients
Hybrid stem cell therapy	Effective for combining the strengths of different stem cell types (e.g., iPSCs for differentiation and MSCs for paracrine support)	Complex to implement and requires co-transplantation of different stem cell types or integration with biomaterials	High cost due to the need for multiple stem cell types and specialized biomaterials	Ensuring the stability and functionality of hybrid stem cell constructs; maintaining consistent results across heterogeneous cell populations
MSC-derived exosomes	Promising in promoting tissue repair and anti-inflammatory responses without the need for live-cell transplantation	Easy to apply in comparison to cell transplantation, but large-scale production and purification can be challenging	Moderate cost, but cell-free nature could reduce long-term treatment costs	Production scalability; ensuring exosome consistency across populations; unknown long-term effects
Activation of developmental signaling pathways	Effective for rejuvenating stem cells and restoring their regenerative potential by reactivating pathways like Wnt, Notch, Hedgehog, and PI3K/Akt	Technically feasible but requires precise control over pathway activation to avoid undesired effects like tumorigenesis	High cost due to the need for specialized reagents and tools for pathway modulation	Excessive or uncontrolled activation of pathways could lead to unwanted effects such as tumorigenesis; requires precise control
3D culture systems	Effective for improving stem cell behavior by providing a more physiologically relevant environment than traditional 2D cultures	Complex and requires specialized equipment and expertise	High initial cost for 3D culture systems, but cost-effective in the long term for large-scale research	Difficulty in translating results from 3D culture systems to in vivo applications; complexity of culture systems
Epigenetic rejuvenation	Effective for resetting the epigenetic clock and restoring stem cell function, particularly through small molecules and histone modifications	Relatively easy to implement, though the long-term effects of epigenetic modulation are not fully understood	Moderate cost for small molecules and inhibitors	Risk of inducing oncogenes or pluripotency; difficulty in achieving precise epigenetic control, especially in heterogeneous cell populations

HPC: Hypoxic preconditioning; ROS: Reactive oxygen species; MSC: Mesenchymal stem cell; iPSCs: Induced pluripotent stem cells; 3D: Three-dimensional; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B.

Hypoxic preconditioning

Hypoxic preconditioning (HPC) is the process by which an organism’s endogenous adaptive mechanisms are activated to develop resistance to subsequent, more severe hypoxic or ischemic insults by exposing it to a period of mild or moderate hypoxia (low oxygen). HPC is one of the most extensively studied strategies to augment stem cell function. In vivo, stem cells naturally reside in hypoxic niches such as bone marrow and adipose tissue, where oxygen tension is significantly lower than atmospheric conditions. Mimicking this physiological hypoxia in vitro by culturing cells under 1%-5% oxygen triggers the stabilization of hypoxia-inducible factors (HIFs), particularly HIF-1α. Stabilized HIFs activate a transcriptional network that promotes angiogenesis (via vascular endothelial growth factor), enhances metabolic adaptation, delays cellular senescence, and sustains stemness. Hypoxia has also been shown to improve mitochondrial efficiency, reduce ROS accumulation, and promote migration and survival of transplanted cells. For elderly stem cells, hypoxic conditioning represents a relatively simple and effective approach to rejuvenate their regenerative potential before therapeutic application. While these stressors can stimulate adaptive mechanisms, they may also increase genomic instability, potentially exacerbating age-related cellular damage. Furthermore, prolonged exposure to hypoxia may lead to the secretion of harmful factors, such as pro-inflammatory cytokines, which could create a detrimental microenvironment, especially in elderly patients. This risk, combined with the heterogeneity in MSC populations, may lead to varying responses to HPC, complicating therapeutic outcomes. The main challenge, therefore, lies in balancing the potential rejuvenating effects of HPC with the risks of inducing further cellular stress, particularly in aged stem cells, and translating these results from in vitro models to in vivo conditions, where oxygen tensions may vary significantly and the long-term effects of HPC remain poorly understood.

GM

GM, also referred to as genetic engineering, is a procedure that involves the modification of an organism’s DNA to introduce new or improve existing traits. It can involve the addition, deletion, or modification of specific genes within an organism’s genome through the use of laboratory technologies. Genetic engineering technologies provide powerful means to reverse age-associated molecular changes in stem cells. GM represents another powerful tool to restore the functionality of aged stem cells. Advanced techniques such as CRISPR/Cas9 allow precise regulation of genes that control differentiation, self-renewal, resistance to oxidative stress, and cellular aging. The innovative CRISPR-Cas genome editing technology has the potential to revolutionize regenerative medicine and aging research by restoring the vitality of elderly stem cells. The regenerative capacity of stem cells decreases as they age due to the accumulation of genetic and epigenetic alterations. CRISPR-Cas can directly modify aging-related pathways or edit these changes, potentially restoring or augmenting cellular function. By enhancing stem cell health and function, this technology has the potential to treat age-related diseases, thereby improving the quality of life of elderly people and establishing new therapeutic pathways. CRISPR technology offers a promising approach to extend the functional lifespan of stem cells and combat cellular aging by targeting aging-related genes. It is possible to remove or change genes that cause senescence, like those in the p16INK4a-pRB and p53-p21 pathways, which are important for controlling the cell cycle and senescence. Editing these critical aging-related genes can delay the onset of stem cell senescence and improve their regenerative capacity and vitality. This method has the potential to substantially extend the functional lifespan of stem cells and offer broad therapeutic implications for age-related diseases and conditions, thus promoting healthier aging and increasing overall lifespan. Even though GM techniques show great promise, challenges remain in ensuring the precision and safety of gene editing, as off-target effects or unintended genetic alterations could undermine the benefits of these interventions. Moreover, the long-term stability of genetically modified stem cells, particularly in clinical settings, needs further investigation.

Growth factor supplementation

Growth factors are proteins that regulate cell growth, differentiation, and survival. Supplementing stem cell cultures with exogenous growth factors can improve the performance of aged cells. Growth factors and cytokine preconditioning can also enhance the therapeutic potential of stem cells by reducing senescence and apoptosis, while simultaneously enhancing self-renewal, cell survival, paracrine activity, and differentiation potential. Growth factors affect several aspects of cells by interacting with their receptors and initiating different downstream signaling cascades. Stem cells, particularly MSCs, release a variety of growth factors and cytokines that affect the cells and tissues in an autocrine or paracrine manner. Insulin-like growth factor promotes survival and differentiation, fibroblast growth factor promotes proliferation and angiogenesis, epidermal growth factor enhances migration and wound repair, and transforming growth factor-β modulates stem cell lineage commitment and extracellular matrix remodeling. These factors promote enhanced tissue healing and mitigate the reduced responsiveness of aged cells during both in vitro expansion and in vivo applications by recreating aspects of a youthful microenvironment. However, the primary limitation of this approach is the short half-life of many growth factors, which limits their long-term therapeutic effects. Additionally, the challenge of controlling the delivery and dosage of these factors in vivo remains a significant barrier to their clinical application. The heterogeneity of MSC populations may also result in differential responses to growth factor supplementation, complicating the consistency of therapeutic outcomes in clinical settings.

Bioactive compounds

Bioactive compounds are substances that biologically react and trigger a response in living tissue, which shows their biological activity. These compounds are present in foods such as fruits, vegetables, and legumes. These compounds can be extracted and purified for a variety of clinical applications. They can promote health or prevent disease, despite not being essential nutrients. Natural and synthetic bioactive molecules are gaining attention as pharmacological agents capable of rejuvenating stem cells without genetic manipulation. Compounds such as resveratrol, curcumin, metformin, and quercetin target pathways involved in oxidative stress response, mitochondrial homeostasis, and autophagy. For example, resveratrol activates sirtuin 1, improving mitochondrial efficiency and delaying senescence, while metformin activates AMP-activated protein kinase, promoting autophagy and metabolic balance. Curcumin and quercetin further modulate mammalian target of rapamycin signaling, reducing age-associated inflammation and preserving stem cell function. Bioactive compounds provide a clinically feasible and non-invasive feasible strategy to augment the therapeutic efficacy of aged stem cells. However, these compounds often require high doses to be effective, and the challenge lies in identifying the optimal therapeutic windows and determining the long-term safety and efficacy of these bioactive agents in stem cell therapies.

Hybrid stem cell therapy

Hybrid stem cell therapy involves combining different stem cell types or pairing cells with biomaterials. It is an emerging approach designed to overcome the limitations of single-cell treatments, such as reduced proliferative or differentiation potential in aged stem cells. One example is the co-transplantation of iPSCs and MSCs, where iPSCs contribute broad differentiation potential and MSCs provide strong paracrine and immunomodulatory support. This combination results in synergistic benefits, including improved survival, enhanced regeneration, and accelerated functional recovery. Similarly, stem cells embedded in biomaterial scaffolds or combined with growth factors provide both structural and biochemical cues, mimicking natural tissue and boosting regenerative outcomes. The difficulty, however, is in maintaining the stability and functionality of hybrid stem cell constructs while optimizing the biomaterial properties to effectively support stem cell behavior in vivo.

MSC-derived exosomes

Exosomes derived from MSCs have emerged as a promising alternative to direct cell transplantation. MSC-derived exosomes can augment the regenerative potential of aged stem cells by providing a cell-free therapeutic approach to deliver bioactive molecules like RNAs, lipids, and proteins that promote cell growth, tissue repair, and anti-inflammatory responses, ultimately revitalizing aged cellular environments. While young stem cells are more potent, using their secreted exosomes offers advantages such as increased stability, longer preservation, and a lack of immunogenicity and potential for neoplastic transformation, making them a promising alternative for cell-free therapies and drug delivery. Importantly, exosomes confer therapeutic benefits without the risks of live-cell transplantation, such as immune rejection or tumorigenicity, while still retaining the regenerative and immunomodulatory properties of their parent MSCs. However, challenges remain in the large-scale production and purification of exosomes, as well as in understanding the long-term effects and potential risks associated with their use, particularly in chronic applications. The heterogeneity of exosome content from MSC populations can also complicate their consistency and effectiveness in therapeutic applications.

Activation of developmental signaling pathways

One of the most promising approaches to rejuvenate aged stem cells lies in the targeted reactivation of fundamental developmental signaling pathways that orchestrate cell fate, growth, and repair. Pathways such as Wnt/β-catenin, Notch, Hedgehog, and phosphatidylinositol 3-kinase/protein kinase B serve as central regulators of stem cell self-renewal, differentiation, and survival. During youth, these signaling cascades operate in a finely balanced manner, ensuring that stem cells remain responsive and capable of repairing tissues. However, with advancing age, these pathways often become dampened, dysregulated, or silenced, contributing to the gradual loss of regenerative potential. By selectively reactivating these signals, researchers are now exploring ways to restore vitality to aged stem cells. For instance, activation of Wnt/β-catenin signaling has been shown to enhance the osteogenic differentiation of MSCs, offering therapeutic opportunities for age-related bone loss such as osteoporosis. Similarly, stimulation of Notch signaling supports the self-renewal of hematopoietic stem cells (HSCs), which is vital for maintaining healthy blood and immune systems in elderly individuals. The phosphatidylinositol 3-kinase/protein kinase B pathway safeguards stem cells against apoptosis and stress-induced damage, thereby improving their survival in hostile or diseased tissue environments. Hedgehog signaling, known for its developmental roles, also contributes to the repair of injured tissues when appropriately reactivated. The tools to achieve such reactivation are diverse and advancing rapidly. Pharmacological agents, small molecules, and growth factors can be used to transiently stimulate these cascades, while gene-editing technologies such as CRISPR provide precise control over the expression of pathway regulators. Importantly, the goal is not simply to turn on these pathways, but rather to restore their youthful balance, as uncontrolled or excessive activation can lead to undesired effects, including tumorigenesis. By reactivating the intrinsic programs that once guided growth and repair, this strategy holds great promise for extending the therapeutic potential of stem cells and developing regenerative treatments for aging-associated disorders. However, activation of these signaling pathways need precise control over pathway activation, as excessive or uncontrolled signaling could lead to tumorigenesis or other undesired outcomes. Moreover, pharmacological agents and gene-editing technologies required to activate these pathways must be further optimized for safety and efficacy.

3D culture systems

Traditional 2D monolayer culture systems fail to replicate the complexity of the in vivo stem cell niche. Emerging 3D culture platforms, including spheroids, organoids, and hydrogel-based scaffolds, provide spatial architecture, mechanical forces, and extracellular matrix interactions that closely mimic physiological conditions. Aged stem cells cultured in 3D systems demonstrate improved viability, enhanced differentiation potential, and greater paracrine activity compared to 2D culture. These systems foster physiologically relevant interactions between stem cells and the extracellular matrix, enhancing their therapeutic utility. However, the complexity of 3D culture systems requires advanced techniques and equipment for proper cell growth, and there are challenges in translating the results from 3D models to in vivo applications, where the heterogeneity of stem cell populations may further complicate outcomes.

Epigenetic rejuvenation

Epigenetic regulation, including DNA methylation, histone modification, and non-coding RNA activity, plays a central role in stem cell aging. Unlike genetic changes, epigenetic alterations are reversible, making them attractive therapeutic targets. Interventions such as small-molecule epigenetic modulators, histone deacetylase inhibitors, and DNA methyltransferase inhibitors can restore youthful epigenetic landscapes, delay senescence, and re-establish multipotency. Moreover, transient expression of reprogramming factors (e.g., octamer-binding transcription factor 4, sex-determining region Y-box 2, Kruppel-like factor 4, cellular myelocytomatosis oncogene) in a controlled manner can partially reset the epigenetic clock without inducing full pluripotency, thereby rejuvenating aged stem cells while minimizing oncogenic risks. The main challenge of epigenetic rejuvenation is to reprogram epigenetic marks in a controlled manner, without triggering unwanted effects like the reactivation of oncogenes or the induction of pluripotency.

CRYOPRESERVATION TO STOP STEM CELL AGING

Cryopreservation is a method used to preserve structurally intact living cells and tissues by cooling them to very low temperatures (Figure 6). It is increasingly regarded not only as a storage technique but also as a powerful anti-aging strategy for stem cells. The fundamental principle behind cryopreservation is that by reducing the temperature of cells to ultra-low levels, typically below -130°C, all biochemical and metabolic activities, including those responsible for cellular aging are effectively suspended. This suspension of biological time stops processes such as telomere shortening, oxidative damage, mitochondrial dysfunction, and epigenetic drift, which are major drivers of age-related decline in stem cell function. In this way, cryopreservation uniquely protects stem cells from progressive senescence, preserving them in a state of youthfulness for many years or even decades[40]. This has profound implications for regenerative medicine, because stem cells harvested and cryopreserved early in life or at their peak functional state can later be thawed and used for therapeutic interventions when regenerative capacity has naturally declined due to aging. Therefore, cryopreservation can be employed as a preventive medical strategy, as well as for storage, to allow individuals to bank their own youthful stem cells as a biological reserve against future degenerative diseases, tissue damage, or organ failure. Patients can take advantage of the strong proliferative potential, differentiation capacity, and paracrine activity of youthful stem cells by reintroducing these preserved cells later in life. This can overcome the restrictions imposed by donor age or age-related decrease. This method is crucial in that it establishes cryopreservation as the cornerstone of future personalized and preventive regenerative medicine by bridging the gap between early stem cell collection and late-life therapeutic requirements[5].

Figure 6 Cryopreservation of cells at slow freezing and rapid cooling. This figure illustrates the two main cryopreservation techniques, slow freezing and rapid cooling. In the slow freezing method, cells are incubated in a cryoprotectant medium with low concentration, leading to some cell damage and extracellular ice formation. In contrast, rapid cooling involves the use of a cryoprotectant medium with high concentration, preserving cell integrity and preventing the formation of ice crystals within the cells.

A successful therapeutic outcome of stem cell-based interventions relies on the timely delivery of viable and functionally competent cells to patients, but logistical challenges frequently arise when the clinical site of application is far removed from cell processing or culture facilities. For some applications, such as MSCs derived from bone marrow, cardiomyocytes from pluripotent stem cells, hepatocytes, peripheral blood stem cells, or alginate-encapsulated adipose-derived stem cells, short-term storage at 4°C can preserve cells for 2-4 days, temporarily bridging these gaps. However, this approach cannot maintain long-term viability, as metabolic disruption and functional decline inevitably occur. In contrast, cryopreservation offers safe, stable, and long-term storage that not only preserves stem cell availability but also slows down their natural aging, making it a critical enabler of regenerative medicine[41]. Despite its transformative role, cryopreservation is not without challenges[42,43]. One of the major challenges is avoiding the formation of ice crystals, which rupture membranes and induce cell death. To address this, cryoprotectants (CPAs) are added to prevent intracellular and extracellular ice formation. Dimethyl sulfoxide (DMSO) is the most commonly used CPA, protecting cells against freezing injury[44]. Initially introduced as an anti-inflammatory agent in medicine, DMSO is still occasionally used to treat autoimmune disorders. It has been shown to be generally safe and non-toxic for stem cells, but it has clinically relevant side effects, including nausea, vomiting, abdominal pain, hemolytic and hepatotoxic effects, as well as renal, central nervous system, cardiovascular, and respiratory complications[45]. In the context of elderly patients who may have compromised organ function or pre-existing conditions, the risks associated with DMSO are increased. Elderly individuals may be more vulnerable to these adverse effects, particularly regarding renal and hepatic toxicity, as well as potential cardiovascular complications. Therefore, careful consideration of the dosing and potential risks of DMSO is essential when using cryopreservation techniques in aged patients, who may have a reduced capacity to metabolize or clear such substances efficiently. Alternative CPAs include propylene glycol, α-tocopherol, ascorbic acid with catalase, and the glucose dimer trehalose, which can act intra- and extracellularly[45]. However, these agents can also introduce adverse effects, such as oxidative stress, membrane permeability issues, and metabolic alterations. Cryopreservation may additionally affect the expression and activity of genes, proteins, and signaling pathways impacting stem cell functionality. Thus, functional and molecular characterization of MSCs before and after cryopreservation, as well as after recovery, is essential. Studies have shown that MSCs can regain functional potency after a 24-hour acclimation period post-thaw. Strategies to further enhance MSC activity and potency include supplementing freezing or culture media with antioxidants, growth factors, or apoptosis/senescence inhibitors[42,43].

Cryopreservation has also revolutionized the use of HSCs, which otherwise rapidly lose viability within hours to days after collection, thereby restricting their use to local and immediate applications. In the case of allogeneic transplantation, fresh HSCs must be infused within 72 hours of collection, a limitation that hinders global donor access. By enabling long-term storage and safe transport of frozen HSCs, cryopreservation expands geographic access, increases genetic donor diversity, ensures rigorous quality testing before transplantation, and has become an essential part of nearly all autologous and allogeneic stem cell transplant procedures. Clinical experience demonstrates that cryopreserved HSCs retain safety and functionality, with no major adverse effects on engraftment or risk of graft-vs-host disease[46,47]. An equally critical application is in UCB banking, where cryopreservation is indispensable because samples are collected at birth and stored for use by either unknown recipients in public banks or by donors and families in private banks, sometimes for indefinite periods. Public cord blood banks, often nonprofit, connect recipients with donors through global registries, while private banks secure UCB for personalized use[48]. Effective cryopreservation is widely recognized as important enabling technology for successful delivery of autologous cell therapies to patients. However, there is a higher risk of decreased engraftment success and cell viability when the samples are preserved far from the collection site or in conditions where travel restrictions or transport unpredictably delays cryopreservation. This emphasizes the need for managing clinical settings so that cryopreservation can be performed timely at the sample collection site. The continued progress of successful cell therapies in regenerative medicine necessitates the development of innovative cryopreservation procedures in the near future.

FUTURE DIRECTIONS

Stem cell-based therapies, particularly those involving MSCs, hold great promise for regenerative medicine, especially in addressing age-related degenerative diseases. While autologous stem cell therapy offers clear advantages, including a reduced risk of immune rejection, optimization of autologous stem cell-based therapies, especially for elderly individuals, demands a comprehensive framework that addresses the interplay between donor age, disease status, niche factors, and rejuvenation strategies. Stem cells from older individuals inherently face reduced proliferative, differentiation, and regenerative potential due to the biological effects of aging, including telomere shortening, mitochondrial dysfunction, and increased ROS. Additionally, age-related diseases such as cardiovascular conditions and diabetes exacerbate the decline in stem cell functionality, making it critical to develop targeted strategies that not only rejuvenate aged stem cells but also account for the complex biological challenges posed by these diseases. The stem cell niche plays a significant role in creating the specialized microenvironment within tissues that supports stem cell function. Aging significantly disrupts the niche, diminishing its capacity to maintain stem cell activity and, consequently, tissue regeneration. Future research should focus on bioengineering the stem cell niche through scaffolds or other tissue engineering methods to rejuvenate these environments, providing a more favorable setting for stem cells to thrive and repair tissues effectively.

To overcome these challenges, a variety of strategies are under investigation, such as HPC, GMs, growth factor supplementation, hybrid stem cell approaches, use of bioactive compounds, MSC-derived exosomes, activation of signaling pathways, 3D culturing, and cryopreservation, are being explored to enhance the therapeutic potential of age-compromised depletion of stem cell function. Additionally, cryopreservation presents a unique opportunity as an anti-aging strategy, allowing stem cells to be preserved in their youthful state and later used for therapeutic interventions when regenerative capacity naturally declines. Integrating these rejuvenation strategies into a unified approach, alongside optimizing the stem cell niche and targeting disease-specific rejuvenation protocols, forms a critical roadmap for future research. This integrated approach ensures that stem cells derived from elderly individuals are as effective in therapeutic settings as those from younger donors, overcoming the limitations imposed by both aging and chronic disease. This framework emphasizes the need for multi-faceted interventions, ranging from genetic engineering to environmental manipulation, to enhance the regenerative potential of stem cells in the aging population. While these approaches have shown promise in restoring or enhancing stem cell function, we are only at the beginning of understanding their complexity and long-term consequences. Critical concerns remain, including the possibility that such interventions may induce toxicity, accelerate senescence, or deplete stem cell reserves elsewhere in the body over time. It is therefore imperative to conduct rigorous clinical trials to evaluate and test the safety, efficacy, and success of these emerging methods. Ultimately, optimizing autologous stem cell therapies requires careful consideration of donor factors and continued development of innovative techniques to maximize treatment efficacy for aged patients.

CONCLUSION

Autologous stem cell therapy offers immense potential for treating age-related degenerative disorders. However, its success is limited by the intrinsic decline in regenerative potential of aged and diseased stem cells. Aging affects stem cell quantity, quality, and responsiveness to their microenvironment, thereby compromising therapeutic outcomes. To address these challenges, innovative rejuvenation approaches such as HPC, GM, growth factor supplementation, bioactive compounds, MSC-derived exosomes, 3D culture systems, and signaling pathway activation have emerged as promising solutions. Cryopreservation stands out as a preventive strategy to preserve youthful stem cells for future therapeutic use. Combination of these strategies with niche engineering and personalized rejuvenation protocols may overcome the age-related limitations of autologous therapies. Carefully designed clinical trials are essential to ensure safety, efficacy, and standardization, which will ultimately lead to the full potential of regenerative medicine for the aged population.

ACKNOWLEDGEMENTS

The authors sincerely acknowledge the valuable suggestions and constructive criticism provided by their colleagues.