Effects of microenvironmental engineering on the stemness of human umbilical cord mesenchymal stem cells

Abstract

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have emerged as highly promising seed cells in regenerative medicine due to advantages such as wide availability, noninvasive collection, strong proliferative capacity, and low immunogenicity. However, the inevitable cellular senescence and loss of stemness during in vitro expansion critically compromise their clinical efficacy. Recent research has shifted focus to the supportive microenvironment. This review elaborates on an emerging strategy, the engineered microenvironment, which aims to effectively maintain and enhance hUC-MSCs stemness by mimicking and manipulating the physical, chemical, and biological signals of the stem cell niche. The article details the key roles and molecular mechanisms of physical factors (e.g., hypoxia, mechanical stimulation, and three-dimensional culture), chemical signals (small molecules and cytokines), and biomaterial scaffolds in regulating hUC-MSCs stemness. It further explores the application potential and enhanced therapeutic efficacy of this strategy in disease models such as bone repair, nerve regeneration, and endometrial repair. Finally, the review outlines challenges, including standardization and scalability. We propose future directions integrating cutting-edge technologies such as artificial intelligence, multi-omics analysis, and smart responsive materials. The aim is to provide new perspectives and a theoretical basis for developing more efficient hUC-MSCs clinical application strategies.

Key Words: Human umbilical cord mesenchymal stem cells; Stemness Microenvironment engineering; Physical signals; Biomaterials; Tissue engineering; Regenerative medicine

Core Tip: Human umbilical cord mesenchymal stem cells (hUC-MSCs) face stemness loss during in vitro expansion, limiting therapeutic efficacy. This review highlights the engineered microenvironment as a pivotal strategy to overcome this bottleneck. By recapitulating the stem cell niche via physical (hypoxia, three-dimensional architecture, and mechanics), chemical (small molecules and cytokines), and biomaterial signals, stemness can be effectively preserved. These approaches enhance hUC-MSCs proliferation, multipotency, and paracrine function, boosting their regenerative potential in disease models. This review offers a theoretical foundation for advancing standardized, microenvironment-based strategies to optimize hUC-MSCs for clinical translation in regenerative medicine.

INTRODUCTION

Mesenchymal stem cells (MSCs) are derived from the mesoderm and have the self-renewal capacity and multi-directional differentiation potential of adult stem cells. They can be differentiated into cartilage, bone, skeletal muscle, and other cell types under certain conditions[1]. MSCs can be isolated from a number of diverse sources and exhibit multi-directional differentiation in vitro. They demonstrate great therapeutic potential in regenerative medicine[2]. For instance, human umbilical cord MSCs (hUC-MSCs) ameliorate liver fibrosis through their immunomodulatory and paracrine mechanisms, highlighting their broad applicability in treating chronic diseases[3]. hUC-MSCs are regarded as an ideal cell source for clinical applications with competitive advantages. They are capable of self-renewal and differentiation, are easily accessible by noninvasive sampling, and have a higher proliferation rate and better self-renewal capacity than cells derived from adult tissues[4]. Stemness pertains to the intrinsic ability of MSCs to undergo self-renewal and differentiate into multiple lineages, while simultaneously impeding their differentiation and preserving crucial differentiating genes in a state of quiescence and equilibrium[5]. Stemness is a dynamic property that is subject to modulation by factor, including cell size, stiffness, and exogenous cues such as oxygen levels. Loss of stemness in MSCs results in an inability to differentiate into diverse cell lineages. This compromises their capacity for tissue and organ regeneration and limits their therapeutic efficacy in various diseases. Stemness is the biological basis for the success or failure of treatment. However, a critical clinical bottleneck is that hUC-MSCs cultured in conventional two-dimensional (2D) in vitro systems exhibit progressively diminished proliferative capacity, reduced differentiation potential, and cellular senescence (loss of stemness) with increasing passages[6]. This phenomenon leads to low post-transplantation cell survival rates, impaired functionality, and compromised therapeutic efficacy.

Conventional approaches have primarily focused on supplementing culture media with growth factors or implementing genetic modifications. However, these strategies are often associated with limitations such as high costs, safety concerns, and inconsistent outcomes. In recent years, accumulating evidence has demonstrated that stem cell fate is precisely regulated by their niche - a specialized microenvironment[7]. This niche constitutes a complex network comprising the extracellular matrix (ECM), neighboring cells, physical and mechanical cues, soluble factors, and metabolic conditions[8,9]. Inspired by this paradigm, the “engineered microenvironment” strategy has emerged. This approach involves the deliberate design and construction of biomimetic in vitro culture systems that recapitulate key features of the in vivo stem cell niche, thereby making the cells into maintaining a more juvenile and functional state[10]. This review aims to systematically summarize recent advances in engineered microenvironments for regulating hUC-MSCs stemness. It will provide an in-depth discussion of the underlying mechanisms from physical, chemical, and biomaterial perspectives, and offer insights into future clinical translation prospects.

STEMNESS CHARACTERISTICS OF HUC-MSCS AND THEIR DEPENDENCE ON THE MICROENVIRONMENT

The umbilical cord is considered medical waste, and the collection of hUC-MSCs is noninvasive. Access to hUC-MSCs is unencumbered by ethical concerns[11]. The immunosuppressive effects have become the most popular property of MSCs for potential clinical use. Many studies have been published about the immunosuppressive effects of umbilical cord Wharton’s jelly’s-MSCs[12], umbilical cord-lining MSCs[13], human umbilical cord perivascular[14], and whole umbilical cord-derived MSCs[4]. These cells exert potent immunomodulatory effects by suppressing T-cell proliferation, modulating dendritic cell maturation, and promoting regulatory T cell expansion. The effects occur largely through paracrine secretion of factors such as prostaglandin E2, indoleamine 2,3-dioxygenase, and transforming growth factor-β (TGF-β)[4,12-14].

hUC-MSCs have a distinct self-renewal capacity while maintaining their multipotency (the ability to differentiate into adipocytes, osteocytes, chondrocytes, neurons, and hepatocytes under appropriate induction conditions)[15,16]. This broad differentiation potential, combined with their robust proliferative capacity, positions hUC-MSCs as highly promising candidates for regenerative medicine applications. Their stemness is characterized by high expression of core pluripotency transcription factors [e.g., octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2)][11], as well as other stemness-associated markers including REX1, LIN28, and c-MYC. Although the expression levels are lower than those in embryonic stem cells, they are significantly higher than those in most adult tissue-derived MSCs[17,18], contributing to their superior expansion capability and differentiation potential.

Single-cell RNA sequencing has revealed considerable heterogeneity among hUC-MSCs, with only a subset (approximately 10%-15%) exhibiting strong stemness and clonogenic capacity[19,20]. Wang et al[19] identified three subpopulations in culture-expanded umbilical cord mesenchymal stem cells (UMSCs), with UMSC1 exhibiting the highest proliferative capacity and stemness. This high-stemness subpopulation is characterized by the expression of proliferation markers including MKI67, TPX2, TOP2A, HMGA2, and HMGB2, as well as cell surface markers such as HMMR and TNFRSF12A. The latter has been proposed as a potential marker for isolating highly proliferative UMSCs. Zhang et al[20] identified a stem-like active proliferative cell subpopulation in MSCs, marked by NG2/CSPG4, CD146/MCAM, and NES, which exhibited high expression of self-renewal-associated transcription factors including E2F1, EZH2, and MYBL2. This heterogeneity manifests as distinct subpopulations varying in the expression profiles of stemness markers, differentiation potential, and secretory capacity. Zhang et al[20] identified two main subpopulations of uncultured hUC-MSCs through single-cell RNA sequencing analysis. Group 1 MSCs were enriched in genes related to immune regulation and oxidative stress response, while group 2 MSCs showed enhanced expression of ECM production and osteochondral differentiation genes[2]. Niche signals are critical determinants of the fate of these subpopulations, influencing their functional specialization and therapeutic potential[19].

The stemness of hUC-MSCs is typically microenvironment-dependent. In vivo, these cells reside within a homeostatic niche characterized by physiological hypoxia (1%-5% O₂), specific cell-cell interactions, and a supportive ECM rich in collagen, fibronectin, and hyaluronic acid[9,21]. This specialized microenvironment provides not only structural support but also essential biochemical and biophysical cues that maintain stem cell quiescence and functionality. Upon isolation and subsequent standard 2D culture, the cells are exposed to non-physiological signals such as atmospheric oxygen tension (21% O₂), rigid planar substrates, and the absence of native three-dimensional (3D) architecture. These cues can trigger differentiation programs or induce cellular senescence through oxidative stress, cytoskeletal tension alterations, and disruption of cell-ECM interactions[9,22]. Studies have demonstrated that conventional 2D culture leads to progressive loss of stemness markers, reduced telomerase activity, and diminished differentiation capacity over successive passages[6,23].

The essence of engineering microenvironments lies in reconstructing or mimicking the physiologically relevant signals of the native stem cell niche, thereby delaying or reversing the attrition of stemness induced by in vitro culture. This approach recognizes that stem cell fate is governed not only by intrinsic genetic programs but also by extrinsic environmental cues that collectively determine cellular behavior and therapeutic efficacy[7-10] (Figure 1, Table 1).

Figure 1 Factors influencing human umbilical cord mesenchymal stem cell stemness.

Table 1 Comparison of the ex vivo culture microenvironment and native in vivo microenvironment of human umbilical cord mesenchymal stem cells.

Feature	In vivo native microenvironment	Ex vivo conventional culture	Consequences of ex vivo deviation
Oxygen tension	Physiological hypoxia (1%-5% O2)[21,22]	Atmospheric normoxia (21% O2)[22,23]	Oxidative stress, accelerated senescence, loss of stemness markers
ECM	Native 3D ECM rich in collagen, fibronectin, and hyaluronic acid; provides biochemical and topographical cues[9,21]	Rigid 2D plastic surface; absence of native ECM composition and architecture[6,9]	Disrupted cell-ECM interactions, altered mechanotransduction, spontaneous differentiation
Cell-cell interactions	Extensive cell-cell contacts within a 3D niche; paracrine signaling networks[8,19]	Limited to 2D monolayer contacts; reduced cell-cell communication[24,25]	Altered signaling pathways, reduced stemness maintenance
Mechanical cues	Soft tissue-specific stiffness (e.g., 0.1-1 kPa for soft tissues); dynamic mechanical forces[9,29]	Supraphysiological stiffness (GPa range of plastic); static, non-physiological substrate[9,29]	Activation of differentiation pathways (e.g., Yes-associated protein/transcriptional coactivator with PDZ-binding motif), loss of multipotency
Biochemical signals	Complex niche-derived soluble factors (growth factors and cytokines) from neighboring cells[7,8]	Simplified culture media; often supplemented with xenogeneic serum (fetal bovine serum)[34,35]	Altered signaling profiles, reduced immunomodulatory capacity
Physical architecture	3D spatial organization with native tissue structure[9,21]	2D planar culture with non-physiological geometry[24,25]	Loss of native cell morphology, altered cytoskeletal organization

ECM: Extracellular matrix; 3D: Three-dimensional; 2D: Two-dimensional.

CORE STRATEGIES AND MECHANISMS FOR ENGINEERING THE MICROENVIRONMENT TO REGULATE HUC-MSCS STEMNESS

The stemness (self-renewal capacity, multipotency, and undifferentiated state) of hUC-MSCs is not solely an intrinsic property but is profoundly governed by extrinsic signals from their surrounding microenvironment, or niche[7,8]. Conventional 2D culture on rigid plastic surfaces fails to recapitulate this complex niche, leading to rapid stemness attrition during in vitro expansion[6]. Engineering the microenvironment aims to overcome this limitation by constructing biomimetic systems that provide physiologically relevant physical, chemical, and biological cues. These engineered niches can proactively direct cellular fate by activating key signaling pathways that maintain stemness, delay senescence, and enhance therapeutic functionality[9,10].

ENGINEERING THE PHYSICAL MICROENVIRONMENT

The physical microenvironment, including oxygen tension, spatial architecture, and mechanical forces, plays an essential role in regulating MSC fate. Recreating and mimicking these parameters in vitro is a promising strategy to sustain stemness and optimize MSC-based therapies.

REGULATION OF OXYGEN TENSION: MIMICKING PHYSIOLOGICAL HYPOXIA

Physiological hypoxia (1%-5% O₂), a hallmark of stem cell niches such as the umbilical cord stroma, is a potent suppressor of differentiation and senescence in hUC-MSCs. Culturing these cells under hypoxic conditions consistently yields superior outcomes compared to conventional normoxic culture (21% O₂). Long-term exposure to 3% O₂ significantly enhances the proliferation rate of Wharton’s jelly MSCs, reduces the expression of senescence-associated markers including p16 and p21, and decreases senescence-associated β-galactosidase activity[21,22]. Concurrently, hypoxia robustly upregulates core pluripotency transcription factors such as OCT4, NANOG, and SOX2, along with other stemness-associated genes including REX1 and c-MYC[21]. The primary mediator of these effects is hypoxia-inducible factor 1-alpha, which stabilizes under low oxygen conditions and translocates to the nucleus. There, it dimerizes with hypoxia-inducible factor 1-beta to activate the transcription of genes involved in glycolysis, cell survival, and stemness maintenance. This transcriptional program drives metabolic reprogramming from oxidative phosphorylation to glycolysis, a characteristic feature of many stem cell populations that supports their undifferentiated state and proliferative capacity[21,23].

3D ARCHITECTURE AND MECHANICAL CUES

Transitioning from 2D monolayers to 3D cultures restores critical cell-cell and cell-ECM interactions, fundamentally altering cell behavior. 3D culture systems, such as spheroids or microencapsulation within biomaterials like alginate, are powerful tools to preserve hUC-MSCs stemness. Compared to conventional 2D monolayers, 3D-cultured hUC-MSCs exhibit significant upregulation of stemness-related genes and enhanced differentiation potential toward osteogenic, chondrogenic, and adipogenic lineages[24,25]. Mechanistically, this functional improvement is often attributed to the activation of key developmental signaling pathways, including the Wingless-related integration site/β-catenin and Hedgehog pathways[26]. Beyond enhancing intrinsic stem cell properties, 3D architectures profoundly reshape the cellular secretome, leading to increased secretion of pro-angiogenic factors (e.g., vascular endothelial growth factor and hepatocyte growth factor) and immunomodulatory molecules. In pre-clinical disease models such as intrauterine adhesion and thin endometrium, 3D-cultured or microencapsulated hUC-MSCs demonstrate superior therapeutic efficacy. This is characterized by enhanced in vivo retention, improved tissue regeneration, and greater functional restoration compared to their 2D-cultured counterparts[27].

Beyond 3D architecture, the physical properties of the substrate itself, including surface topography and stiffness, provide potent mechanical cues that regulate hUC-MSCs fate. Nanofibrous scaffolds engineered from materials such as polyethersulfone or chitosan closely mimic the fibrous structure of the native ECM. By presenting nanoscale topological features, these scaffolds promote a more in vivo-like cell morphology, enhance the expression of stemness markers, and guide lineage specification. This process is initiated by the clustering of integrin receptors, which triggers cytoskeletal reorganization and activates mechanotransduction signaling pathways that ultimately influence gene expression in the nucleus[9,28]. Similarly, hUC-MSCs are exquisitely sensitive to substrate stiffness, a mechanical property sensed through actomyosin contractility. Culture on soft hydrogels that recapitulate the elasticity of brain or adipose tissue (approximately 0.1-1 kPa) tends to maintain stemness, whereas rigid substrates mimicking the mechanical environment of bone (approximately 25-40 kPa) potently drive osteogenic differentiation. This stiffness-sensing mechanism is primarily mediated by the translocation and activation of the transcriptional co-activators Yes-associated protein and transcriptional coactivator with PDZ-binding motif, which serve as downstream effectors of the mechanical signal[9,29].

In addition to static substrate properties, dynamic physical stimuli such as electrical fields and magnetic forces are effective noninvasive tools for modulating hUC-MSC fate. Culturing MSCs on piezoelectric materials like β-poly(vinylidene fluoride) films provides a unique bioactive platform. These materials generate transient micro-scale electrical potentials (approximately 1.5-2.0 V) in response to mechanical stress, creating a dynamic electrophysiological microenvironment. When cultured on these piezoelectric substrates for 7-14 days, MSCs expanded on such piezoelectric substrates exhibit marked delay in cellular senescence. This is evidenced by the maintenance of longer telomeres and sustained high expression of pluripotency markers, including OCT4 and NANOG. This anti-senescence effect is likely mediated through the mitigation of oxidative stress, leading to enhanced in vivo wound healing capacity[30]. Similarly, exposure to static magnetic fields (SMFs), such as those generated by samarium cobalt with a magnetic flux density of 0.2-0.3 T, promotes hUC-MSCs proliferation. Continuous exposure to SMFs for 3-7 days significantly increases the proportion of cells in the S and G2/M phases of the cell cycle and concurrently upregulates pluripotency markers. These findings suggest that SMF treatment represents a promising noninvasive strategy to boost stem cell expansion ex vivo while effectively preserving their core stemness properties[31] (Figure 2).

Figure 2 Physical factors modulating the stemness of human umbilical cord mesenchymal stem cells. YAP: Yes-associated protein; TAZ: Transcriptional coactivator with PDZ-binding motif; Wnt: Wingless-related integration site; HIF-1α: Hypoxia-inducible factor 1-alpha; HIF-1β: Hypoxia-inducible factor 1-beta; OCT4: Octamer-binding transcription factor 4; NANOG: Nanog homeobox; SOX2: SRY-box transcription factor 2; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Engineering the chemical microenvironment

The biochemical composition of the niche, including small molecules, growth factors, and extracellular vesicles, provides precise instructions for stem cell fate. Small molecules offer a powerful and controllable approach to modulate key signaling pathways that govern MSC fate. Selective inhibition of the TGF-β pathway using the receptor inhibitor SB431542 enhances the osteogenic potential of induced pluripotent stem cell-derived MSCs while reducing premature senescence during differentiation, highlighting how targeted pathway inhibition can steer MSC progeny toward a more therapeutically robust phenotype[7,32]. Glucocorticoid preconditioning with dexamethasone, at optimized concentrations, upregulates core stemness markers (OCT4, SOX2, NANOG, and Krüppel-like factor 4). It also upregulates potent immunomodulatory molecules such as prostaglandin E2 and indoleamine 2,3-dioxygenase in various tissue-specific MSCs, including those derived from the umbilical cord. This effect is primarily mediated through activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, effectively priming the cells for enhanced immunosuppressive function upon transplantation[33]. Melatonin pretreatment improved the survival and therapeutic efficacy of hUC-MSCs in a type II diabetes mouse model. This beneficial effect is achieved through modulation of macrophage immunomodulation and activation of the PI3K/AKT pathway in recipient tissues, leading to improved glycemic control and insulin sensitivity[34].

Beyond the use of defined small molecules, the composition of culture supplements plays a critical role in maintaining stemness, particularly as the field moves toward clinical translation. Transitioning from conventional fetal bovine serum to human-derived supplements is essential to eliminate the risks of xeno-immunization and pathogen transmission. Human cord blood serum (CBS) and platelet lysate (PL) represent two promising alternatives, as they are rich in a complex mixture of growth factors (including platelet-derived growth factor, TGF-β, and insulin-like growth factor) and cytokines. Accumulating evidence indicates that CBS is particularly effective, supporting higher hUC-MSC proliferation rates and better maintenance of stemness and multipotency compared to fetal bovine serum[34-36]. PL also induces chondrogenic differentiation of hUC-MSCs, even in the absence of exogenous TGF-β, underscoring its potent bioactivity and potential for directed differentiation applications[35].

In addition to direct supplementation with biochemical factors, the paracrine signals secreted by cells can be harnessed to precondition MSCs and enhance their therapeutic potential. Platelet-rich plasma-derived exosomes are enriched with growth factors and microRNAs. They significantly boost hUC-MSCs proliferation, viability, migration, and resistance to apoptosis when used as a priming agent. These primed MSCs exhibited markedly enhanced therapeutic efficacy in a peripheral nerve injury model, largely attributed to increased secretion of glial-derived neurotrophic factor mediated by the PI3K/Akt signaling pathway[36]. A particularly intriguing example of how circulating factors influence MSC fate comes from studies of naturally skinny individuals. Alessio et al[37] demonstrated that priming MSCs with sera from skinny people reduced spontaneous adipogenesis and, upon induced differentiation, promoted bias toward brown rather than white adipocyte differentiation. This was evidenced by smaller lipid droplets, increased expression of adipose triglyceride lipase, higher mitochondrial content, and upregulation of UCP1 (all hallmarks of brown adipocytes). These findings provide insights into the physiopathology of obesity and highlight the profound impact of circulating factors on MSC lineage specification. They reinforce the concept that the chemical microenvironment, whether engineered or naturally occurring, can direct stem cell fate. In a complementary approach, conditioned medium collected from hUC-MSCs cultured under optimized conditions, such as with CBS supplementation, has an enhanced secretome profile. This optimized conditioned medium can promote proliferation and wound healing in other cell types, highlighting the broader potential of using engineered secretomes for cell-free therapeutic strategies[38].

BIOMATERIALS AND CONSTRUCTION OF COMPOSITE MICROENVIRONMENTS

Biomaterials are the structural foundation for engineered niches, providing mechanical support and biochemical signaling to regulate hUC-MSCs fate. Among naturally derived biomaterials, collagen and chitosan have particular promise for stemness maintenance. An injectable collagen hydrogel combined with hUC-MSCs was shown to prolong cell retention in the uterine cavity and upregulate the expression of core stemness genes, including OCT4, NANOG, and SOX2. This approach improved endometrial regeneration and fertility recovery in a rat model of thin endometrium, underscoring the therapeutic potential of combining biomaterials with stem cells[39]. Similarly, chitosan, a natural polysaccharide, promotes the formation of hUC-MSCs spheroids. When combined with hypoxic culture conditions, chitosan films synergistically enhance the expression of OCT4, SOX2, and NANOG, positioning chitosan as an excellent substrate for stemness maintenance[28].

In parallel with natural materials, synthetic and smart biomaterials offer the advantages of defined composition and tunable properties. Functionalized polymer brushes, such as those grafted with RGD peptides on poly(OEGMA-co-HEMA) substrates, provide a fully defined, xeno-free culture surface that supports long-term self-renewal of human pluripotent stem cells. This principle that can be readily adapted for hUC-MSCs expansion under clinically compliant conditions[29]. Beyond static substrates, smart hydrogels engineered to respond to external stimuli (e.g., temperature, pH, or light) represent an advanced platform for dynamic microenvironment control. These materials can be designed to present or release bioactive factors in a temporally programmed manner, enabling precise regulation of stem cell fate decisions[10].

Perhaps the most biomimetic approach involves the use of decellularized extracellular matrices (dECMs), which maximally preserve the complex biochemical and topographical cues of the native stem cell niche. dECM derived from bone tissue retains the native compositional and structural features that support osteogenic commitment. When hUC-MSCs are seeded on decellularized bone matrix, they exhibit upregulated expression of osteogenic markers such as collagen I and osteonectin, even in the absence of osteoinductive supplements. Simultaneously, they display modulated expression of stemness-related genes. These findings demonstrate the potent inductive capacity of native ECM, which provides not only structural support but also biochemical and topographical signals that engage integrin-mediated signaling pathways, activate mechanotransduction cascades, and ultimately influence stem cell fate decisions (including the balance between stemness maintenance and lineage commitment) through physiologically relevant cues[40]. Beyond bone-derived dECM, decellularized Wharton’s jelly matrix from the human umbilical cord preserves the native composition of the Wharton’s jelly niche. This provides a homologous microenvironment that supports stem cell phenotype maintenance, evidenced by the preservation of primitive markers and enhanced self-renewal capacity[41]. Similarly, decellularized endometrial ECM enhances the adhesion, proliferation, and clonogenicity of tissue-resident MSCs while maintaining stemness marker expression[42]. These findings underscore the versatility of tissue-specific dECMs as a platform for engineering customized microenvironments tailored to diverse regenerative applications.

APPLICATIONS AND FUTURE PROSPECTS: ENGINEERING MICROENVIRONMENTS TOWARD CLINICAL TRANSLATION

Engineered microenvironment strategies have demonstrated the potential to enhance the therapeutic efficacy of hUC-MSCs in various disease models. In bone defect models, induced pluripotent stem cell-derived human MSCs treated with TGF-β inhibitors or hUC-MSCs cultured on osteogenic biomimetic materials exhibited enhanced osteogenic capacity[32,43]. In a peripheral nerve injury model, hUC-MSCs preconditioned with platelet-rich plasma-derived exosomes significantly promoted axonal regeneration and functional recovery via heightened secretion of glial-derived neurotrophic factor[36]. 3D culture-derived exosomes from human embryonic stem cells inhibit hepatic stellate cell activation, thereby ameliorating liver fibrosis[44]. In situ transplantation of 3D hUC-MSCs spheroids more effectively ameliorates premature ovarian insufficiency compared to monolayer-cultured hUC-MSCs. This enhanced efficacy is mediated through paracrine actions that significantly mitigate oxidative stress, consequently preventing apoptosis and autophagy in ovarian granulosa cells[45].

Despite the promise of engineered microenvironments for hUC-MSCs expansion, several critical challenges must be addressed before widespread clinical translation. Foremost among these is standardization, as optimal parameters (including precise hypoxia concentrations, mechanical cue intensities, and effective small molecule combinations) have yet to be unified across different studies and laboratories. This lack of consensus is compounded by challenges in scalable production, as it remains technically demanding to adapt complex 3D culture systems or material-based processing methods to meet the rigorous demands of clinical-grade cell manufacturing under Good Manufacturing Practice standards. Our mechanistic understanding of how microenvironmental signals are transduced across the cell membrane and ultimately influence epigenetic modifications and gene expression programs remains incompletely elucidated, limiting our ability to rationally design optimized culture systems.

Several emerging technological directions hold promise for overcoming these limitations. The integration of high-throughput screening platforms with artificial intelligence offers a powerful approach to systematically explore the vast combinatorial space of microenvironmental parameters, enabling rapid identification of optimal conditions for stemness maintenance. Concurrently, developing dynamic and personalized microenvironments through smart materials capable of responding to cell states or external stimuli, such as light and magnetic fields, will enable more precise, temporally controlled regulation of stem cell fate. Finally, the continued advancement of organoids and organ-on-a-chip technologies will allow investigation of hUC-MSCs stemness regulation within increasingly complex in vitro bionic systems, such as a dedicated MSC niche-on-a-chip. Such microfluidic platforms enable precise control of physical and chemical cues (including fluid shear stress, oxygen gradients, and biochemical signals), and real-time visualization of cellular responses. By incorporating multiple cell types (e.g., endothelial and immune cells) and ECM components, these systems can recapitulate key features of the native stem cell niche, enabling high-throughput screening of microenvironmental parameters and providing valuable mechanistic insights into hUC-MSCs stemness regulation under physiologically relevant conditions. These physiologically relevant models promise to provide more reliable predictive insights for in vivo applications, ultimately accelerating the translation of engineered microenvironment strategies from bench to bedside.

Several controversies and ethical considerations also warrant further attention. Single-cell sequencing studies have revealed considerable heterogeneity within hUC-MSCs populations[19,20,46], yet standardized characterization criteria remain elusive. This highlights the need for a more precise understanding of stemness regulation. The transition from animal-derived (e.g., PL) addresses xeno-immunization concerns but introduces challenges in standardization and batch-to-batch variability, although recent multinational efforts have demonstrated the feasibility of producing standardized human PL across different blood establishments[47]. Ethical considerations regarding umbilical cord tissue sourcing require rigorous informed consent protocols, and contemporary ethical frameworks call for responsible innovation, transparency, and meaningful public engagement[48]. Regulatory frameworks must evolve to balance patient safety with innovation. The International Society for Cell and Gene Therapy emphasizes that standardization of donor factors, manufacturing processes, and clinical trial design is essential for consistent therapeutic outcomes[4,49]. Addressing these controversies and ethical challenges will be essential for successful clinical translation of hUC-MSCs-based therapies.

The clinical translation of hUC-MSCs also faces critical challenges related to potential hazards, inherent limitations, and patient selection. Long-term ex vivo expansion carries a theoretical risk of chromosomal aberrations and malignant transformation, though rigorous manufacturing practices and limited population doublings can mitigate such risks[50]. Key limitations include significant inter-donor heterogeneity, loss of stemness during prolonged culture, and the lack of standardized manufacturing protocols. These factors all contribute to inconsistent clinical outcomes[51]. Microenvironment modulation (e.g., hypoxia, 3D culture, and small molecule preconditioning) directly addresses these limitations by preserving stemness, enhancing in vivo survival, and improving product consistency. Regarding patient selection, hUC-MSCs are particularly well-suited for conditions where immunomodulation and tissue regeneration are the primary therapeutic mechanisms. Clinical studies have demonstrated the efficacy of mesenchymal stromal cells in steroid-refractory acute graft-vs-host disease[52] and complex perianal fistulas in Crohn’s disease[53], highlighting their potential in immunoinflammatory disorders. Preclinical evidence supports their use in endometrial injury, bone defects, and peripheral nerve damage[36,39]. Addressing these hazards, limitations, and patient selection criteria through optimized microenvironment engineering is essential for the safe and effective clinical use of hUC-MSCs.

CONCLUSION

Engineering the in vitro microenvironment for hUC-MSCs represents an effective and promising strategy to address loss of stemness in clinical applications. By integrating physical, chemical, and biomaterial signals to mimic the physiological stem cell niche, this approach significantly enhances the proliferation, stemness maintenance, and therapeutic function of hUC-MSCs. This interdisciplinary field, which integrates materials science, biomechanics, molecular biology, and clinical medicine, holds great potential to advance the safe and efficient application of hUC-MSCs in regenerative medicine. Ultimately, this will improve human health.