Human Bone Marrow-Derived Mesenchymal Stem Cell Applications in Neurodegenerative Disease Treatment and Integrated Omics Analysis for Successful Stem Cell Therapy

Neurodegenerative diseases (NDDs), which are chronic and progressive diseases, are a growing health concern. Among the therapeutic methods, stem-cell-based therapy is an attractive approach to NDD treatment owing to stem cells’ characteristics such as their angiogenic ability, anti-inflammatory, paracrine, and anti-apoptotic effects, and homing ability to the damaged brain region. Human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) are attractive NDD therapeutic agents owing to their widespread availability, easy attainability and in vitro manipulation and the lack of ethical issues. Ex vivo hBM-MSC expansion before transplantation is essential because of the low cell numbers in bone marrow aspirates. However, hBM-MSC quality decreases over time after detachment from culture dishes, and the ability of hBM-MSCs to differentiate after detachment from culture dishes remains poorly understood. Conventional analysis of hBM-MSCs characteristics before transplantation into the brain has several limitations. However, omics analyses provide more comprehensive molecular profiling of multifactorial biological systems. Omics and machine learning approaches can handle big data and provide more detailed characterization of hBM-MSCs. Here, we provide a brief review on the application of hBM-MSCs in the treatment of NDDs and an overview of integrated omics analysis of the quality and differentiation ability of hBM-MSCs detached from culture dishes for successful stem cell therapy.


Introduction
Neurodegenerative diseases (NDDs) are caused by the progressive degeneration of the structure and function of neurons and glial cells in the central and peripheral nervous systems [1,2]. NDDs can be classified according to their causes and symptoms [3]. Alzheimer's disease (AD) and Parkinson's disease (PD) are among the most common neurodegenerative disorders. AD shows widespread degeneration of several types of neurons, whereas PD shows selective loss of a specific cell population, such as dopaminergic neurons. Amyotrophic lateral sclerosis (ALS), commonly called Lou Gehrig's disease, shows selective degeneration of the upper and lower motor neurons [4] and has been associated with genetic mutations in the enzyme Cu/Zn superoxide dismutase 1 (SOD1) [5,6]. Huntington's disease (HD) is a rare genetic disorder caused by a mutation in the huntingtin gene that causes movement abnormalities and cognitive impairment [7]. As the human lifespan increases, the social burden of NDDs also increases [8]. Due to the incurable and debilitating nature of these conditions, there are several limitations in treating NDDs with conventional methods. Therefore, there is a need for new treatment approaches.
Because MSCs can accelerate tissue regeneration, they are excellent candidates for tissue engineering. Vacanti and Langer define tissue engineering as an interdisciplinary field in which engineering and biological sciences are applied together to develop biological substitutes that can restore, maintain, or enhance tissue function [51]. Tissue engineering facilitates autologous MSCs' transplantation by seeding the patient's cells onto a biodegradable scaffold that forms a specific tissue that can then be used to repair injuries [52]. Despite all this, MSC-based tissue engineering faces some challenges, such as the low survival rates of MSCs and the uncertainty of MSC differentiation after infusion. To address these shortcomings, biomaterials are used together with MSCs to maintain their viability, serve as a substrate for cell adhesion, induce differentiation into specific target cells, and as a mechanical tool for tissue regeneration. Biomaterials have demonstrated excellent biocompatibility, provide a suitable cellular microenvironment for MSCs, and are effective and easy to administer. As biomaterials are diverse in their physical, chemical, mechanical, and biological properties, they can contribute to tissue regeneration in different types of injuries [53]. Although several biomaterials have been developed, hydrogels, nanofibers, carbon-based nanomaterials, and cell-free scaffolds have emerged as the frontrunners. For example, the 3D structure of hydrogels supports the proliferation of cells while acting as a barrier to harmful factors [54]. Yan et al. investigated collagen-chitosan scaffolds with BM-MSCs as a therapeutic strategy for traumatic brain injury. Collagen scaffolds with human MSCs have been shown to improve spatial learning and sensorimotor functions, while chitosan serves as a neuroprotector. They reported that these scaffolds exhibited low immunogenicity, good biocompatibility, and therapeutic effects such as neurological, behavioural, and cognitive recovery [55].
In clinical applications, the quality of hBM-MSCs should be analyzed after cell detachment from the culture dish [12,15,56], because cell quality is an essential factor for therapeutic effects. However, there are limitations to the traditional methods used to evaluate the quality and freshness of cells prior to transplantation of hBM-MSCs into humans, such as trypan blue staining or fluorescence-activated cell sorting (FACS) [56,57]. Regardless, it is possible to increase the treatment effect by determining the exact cell condition using various big data analyses, including omics and bioinformatic analyses of the quality and differentiation ability of hBM-MSCs over time after cell detachment from the culture dish. In this review, the transcriptome and metabolome, analyzed using microarray and gas chromatography-mass spectrometry (GC-MS), respectively, were integrated to evaluate the condition of hBM-MSCs. Integrated omics analysis of hBM-MSCs showed increases in reactive oxygen species (ROS) production, lipid peroxidation, and cell damage, leading to the loss of cell quality and differentiation ability as the phosphate-buffered saline (PBS) storage time increased (Figure 1). In clinical applications, the quality of hBM-MSCs should be analyzed after cell detachment from the culture dish [12,15,56], because cell quality is an essential factor for therapeutic effects. However, there are limitations to the traditional methods used to evaluate the quality and freshness of cells prior to transplantation of hBM-MSCs into humans, such as trypan blue staining or fluorescence-activated cell sorting (FACS) [56,57]. Regardless, it is possible to increase the treatment effect by determining the exact cell condition using various big data analyses, including omics and bioinformatic analyses of the quality and differentiation ability of hBM-MSCs over time after cell detachment from the culture dish. In this review, the transcriptome and metabolome, analyzed using microarray and gas chromatography-mass spectrometry (GC-MS), respectively, were integrated to evaluate the condition of hBM-MSCs. Integrated omics analysis of hBM-MSCs showed increases in reactive oxygen species (ROS) production, lipid peroxidation, and cell damage, leading to the loss of cell quality and differentiation ability as the phosphate-buffered saline (PBS) storage time increased (Figure 1). Figure 1. Effect of storage time on cell quality and differentiation potential. Before transplantation, human bone-marrow-derived mesenchymal stem cells (hBM-MSCs), which were expanded ex vivo and detached from the culture dish, were analyzed to evaluate their quality and differentiation potential. The transcriptome [58] and metabolome [57] were analyzed using microarray and gas chromatography-mass spectrometry (GC-MS), respectively. By integrating these datasets with in silico prediction, it was found that as the quality and differentiation potential of hBM-MSCs decreased and ROS production, lipid peroxidation, and cell damage increased in phosphate-buffered saline (PBS) over time.
Omics-based approaches are actively applied and developed to reflect diverse and complementary biological phenotypes and elucidate precise molecular mechanisms as the availability of high-throughput data technologies increases in the biological and medical fields [59]. These have been beneficial in identifying biomarkers for the diagnosis of various diseases. Nevertheless, most omics analyses have been limited to a single dataset, Figure 1. Effect of storage time on cell quality and differentiation potential. Before transplantation, human bone-marrow-derived mesenchymal stem cells (hBM-MSCs), which were expanded ex vivo and detached from the culture dish, were analyzed to evaluate their quality and differentiation potential. The transcriptome [58] and metabolome [57] were analyzed using microarray and gas chromatography-mass spectrometry (GC-MS), respectively. By integrating these datasets with in silico prediction, it was found that as the quality and differentiation potential of hBM-MSCs decreased and ROS production, lipid peroxidation, and cell damage increased in phosphate-buffered saline (PBS) over time.
Omics-based approaches are actively applied and developed to reflect diverse and complementary biological phenotypes and elucidate precise molecular mechanisms as the availability of high-throughput data technologies increases in the biological and medical fields [59]. These have been beneficial in identifying biomarkers for the diagnosis of various diseases. Nevertheless, most omics analyses have been limited to a single dataset, which is accompanied by difficulty in reflecting the actual phenotype [60]. In addition, the datasets used for computational analysis have advanced from structured one to big Bioengineering 2023, 10, 621 4 of 21 data with various unstructured and semi-structured characteristics, and the relationship between omics data is expected to become more complex [61]. Artificial intelligence (AI) is increasingly essential in big data mining, including the biological and medical fields [62,63]. Among AI, machine learning and deep learning approaches exert tremendous power in processing and modelling vast and diverse omics data [64]. Integrated omics analysis aims to utilize big data, machine learning, and systematic algorithms to obtain patterns between data and make more accurate predictions [65,66]. Convergence with computational science is necessary for this type of analysis [67].
The current review has two sections. In the first section, we provide an overview of studies on the application of hBM-MSCs to the treatment of various NDDs, and in the second section, we provide an integrated omics analysis of the quality and differentiation ability of hBM-MSCs according to PBS storage time for successful stem cell therapy.

Application of hBM-MSCs for the Treatment of Various Neurodegenerative Diseases
Neurodegenerative diseases are characterized by the selective dysfunction and progressive loss of neurons, glial cells, and neural networks in the brain and spinal cord. Synaptic dysfunction, neuronal loss, proteasome dysfunction, and the aggregation of misfolded proteins are common NDD features. NDDs affect multiple facets of function in humans, thereby limiting their ability to perform even the most basic tasks [4,68,69].
Stem cells are crucial for the development, growth, and repair of various tissues and organs. In addition, stem cells have provided breakthroughs across all fields of research and medicine owing to their multipotency and self-renewal properties. MSCs have demonstrated numerous neuroprotective effects such as decreased apoptosis, reduced ROS generation, and the promotion of neuronal growth. In particular, hBM-MSC transplantation has been shown to improve clinical outcomes, decrease cerebral atrophy, and enhance patient performance [4,70,71]. In this section, we briefly review the neuroprotective effects of clinically applied hBM-MSCs on some common NDDs.

AD
AD is an NDD associated with a progressive decline in cognitive and memory functions. The pathological features of AD include the aggregation of amyloid beta peptides (Aβ), forming amyloid plaques, intracellular neurofibrillary tangles, and hyperphosphorylated tau and leading to neuronal death. Continual build-up of Aβ activates microglia, thus accelerating neuronal loss, cognitive decline, tau pathology, and the secretion of proinflammatory cytokines. These factors induce synaptic deficits in the hippocampus, leading to cognitive impairment and memory decline [50,68,71,72]. Conventional AD treatments consist of two types of pharmacological therapies. The first includes the use of cholinesterase inhibitors to relieve physical symptoms by increasing the levels of the neurotransmitter acetylcholine. The second type of therapy uses memantine, a drug that improves symptoms by inhibiting N-methyl-D-aspartate (NMDA) receptors. Although several drugs and natural compounds are available for the treatment of AD, drugs that can prevent or delay the progression of AD are yet to be discovered [72].
Transplanted hBM-MSCs can differentiate into neurons, produce neurotrophic factors such as BDNF and NGF, and inhibit Aβ-and tau-related cell death [71]. Numerous studies have explored the neuroprotective effects of BM-MSCs in AD mouse models. Lee et al. achieved a significant reduction in oxidative stress, improvement in cognitive function, and mitigation of Aβ-induced neuronal injury both in vitro and in vivo after co-culturing BM-MSCs with hippocampal neurons stimulated by Aβ [73]. In addition, hMB-MSC-derived vesicles alleviated cognitive decline, reduced the number of intracellular plaques, decreased chronic inflammation, and delayed AD pathogenesis in a preclinical mouse model [74]. BM-MSC-derived exosomes have also been shown to ameliorate cognitive damage by secreting miRNAs capable of enhancing neuronal plasticity, promoting cell survival and synaptogenesis, and suppressing inflammation [75]. Collectively, these studies highlight the multiple advantages of BM-MSCs as a therapeutic agent for AD.

PD
PD is the most common synucleinopathy characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the accumulation of α-synuclein in Lewy bodies, causing tremors, bradykinesia, and cognitive dysfunction [76]. Dopamine (DA) is a neurotransmitter that transmits information between the SNpc and other parts of the brain, thereby controlling the body's voluntary movements. Mitochondrial dysfunction, excessive ROS generation, and impairment of the ubiquitin-proteasome system are involved in DA neuronal degeneration [77]. A typical therapy for PD involves treatment with the DA precursor L-3,4-dihydroxyphenylalanine (L-Dopa), which can produce adverse effects, such as non-responsiveness and abnormal uncontrollable movements or dyskinesia, upon longterm use. Furthermore, this form of therapy focuses only on alleviating symptoms instead of resolving the primary cause of the disease, thereby permitting disease progression [78,79].
MSC-based cell therapies offer diverse options for the treatment of PD. One case in point involves the implication of a defective autophagy system as a plausible cause of PD. MSCs have been reported to display α-synuclein clearance, the regulation of autophagy lysosomal activity, the activation of autophagy signalling and immunomodulatory effects, such as penetrating injured sites, releasing numerous growth factors, and attenuating inflammation [79]. Another study investigating neural-induced hBM-MSCs (NI-hBMSCs) demonstrated increased cell survival, stabilization of α-synuclein monomers, and promotion of neurogenesis after treatment with NI-hBMSCs [80]. Clinical trials in which hBM-MSCs from healthy donors were intravenously administered to patients with PD have shown promising results. The participants in the study exhibited post-infusion changes in motor and non-motor symptoms that lasted until the end of the study period [81]. Based on the results from these and several other studies on patients with PD and animal models, the therapeutic benefits of hBM-MSCs over conventional treatments are evident.

ALS
ALS is a gradual, fatal, paralytic NDD characterized by the degeneration of the upper and lower motor neurons. ALS causes weakness and atrophy of the muscles of the limbs, chest, neck, and oropharyngeal area and eventually death due to respiratory failure. As there are only two drugs currently approved for ALS treatment, there is an urgent need for different treatment options [82,83]. MSCs are being explored as a treatment option for ALS because they produce and release neurotrophins, which are proteins that induce the survival, development, and function of neurons. Transplantation of hMSCs has also been reported to mitigate neuroinflammation, improve motor execution, and enhance the bioenergetics of recipient cells [84][85][86].
Clinical trials using hBM-MSCs as therapeutic agents for ALS have shown favourable outcomes after hBM-MSC administration. In one open-label phase I trial, TGF-β and IL-10 levels were elevated following hBM-MSCs administration. TGF-β, a growth factor involved in various aspects of neuron development and function, has been found to be reduced in ALS patients and inversely correlated to disease progression [87]. Another open-label study conducted to evaluate the safety and efficacy of autologous hBM-MSCs via intrathecal and intravenous routes in ALS patients demonstrated a temporary decline in ALS progression after a single dose of hBM-MSCs [88]. To maximize the capability of hBM-MSCs to treat ALS, additional studies are needed on the effective delivery of MSCs to patients, the effectiveness of MSCs expressing diverse growth factors, and their clinical significance [89].

HD
HD is an autosomal dominant neurodegenerative disease caused by the loss of gammaaminobutyric acidergic (GABAergic) medium spiny neurons in the striatum. This neuronal loss stems from the expansion of the cytosine-adenine-guanine (CAG) repeat within exon 1 of the huntingtin (htt) gene, which leads to the formation of a malfunctioning mutant HTT protein [90]. The clinical manifestations of HD include chorea, psychiatric symptoms, and cognitive impairment. The reduced availability of neurotrophic factors, such as NGF, BDNF, and neurotrophin-3 (NT-3), contributes to neurodegeneration and therefore, these are considered potential therapeutic agents for HD [91].
In a mouse model of HD using transplanted hBM-MSCs, intrastriatally transplanted hBM-MSCs not only successfully survived and differentiated but also reduced motor function impairment, increased neurogenesis, and boosted animal survival and cell differentiation [92]. Given that levels of neurotrophic factors (NTFs) are reduced in patients with HD, NTF-based therapies are potential strategies for the discovery of new treatment options for HD. Since BDNF has a short half-life, which limits effective delivery strategies for NDDs, genetically engineered hBM-MSCs that deliver BDNF (MSC/BDNF) have advantages of delivering BDNF to the striate and MSC-secreted factor supplementation. Pollock et al. reported a significant surge in neurogenesis, an increased lifespan, and decreased spinal atrophy in mice transplanted with MSCs/BDNF [93]. Moreover, hBM-MSCs induced to differentiate into NTF-secreting cells (NTF + ) exhibit therapeutic properties and attenuate neurotoxicity [94]. Although the therapeutic ability of hBM-MSCs in the treatment of HD has been established, additional studies to accurately determine the administration time, dose, and frequency of cells, as well as long-term toxicology studies, should be conducted.

Analysis of the Quality and Differentiation Ability of hBM-MSCs
To date, many studies on NDDs have shown different features of neurodegeneration, such as cell viability reduction, genetic mutations, gene expression alterations, and cellular function impairment [95][96][97]. To understand the different cellular processes in NDDs, cell health has been evaluated using several methods, including morphological analysis, viability assays, metabolic assays, and gene expression analysis [98][99][100], because cell quality directly affects cell functionality [101,102].
Similarly, because the quality and differentiation potential of stem cells are crucial for their use in NDD therapy, the condition and properties of stem cells should be assessed prior to use [103]. Moreover, BM-MSCs show a loss of stemness when maintained under certain conditions [104,105]. Stolzing et al. reported that the overall hBM-MSC fitness decreased with age, showing an increase in ROS, p21, and p53 levels [104]. These deteriorating features were also observed during in vitro ageing. Geissler et al. showed that progenitor characteristics were lost, and genes related to cell differentiation, focal adhesion organisation, cytoskeleton turnover, and mitochondrial function were downregulated during long-term in vitro expansion of MSCs [105].
Meanwhile, there are some studies suggesting that gender may affect the efficiency of BM-MSC therapy [106,107]. Sammour et al. reported that female BM-MSCs have more therapeutic effects than male BM-MSCs by showing greater pro-angiogenic and anti-inflammatory effects in mice models [106]. In addition, Crisostomo et al. demonstrated that female BM-MSCs showed lower apoptosis, TNF and IL-6 production, and higher VEGF expression upon stress activation than males, due to their inherent resistance to TNFR1 activation [107]. However, one study reported that in vitro mesodermal differential capacity of hBM-MSCs is not highly related to the donor gender [108]. Although donor gender seems to play a role in the therapeutic effects of BM-MSCs, this is still not clearly elucidated and further studies are required to clarify the effect of gender on BM-MSCs.
Even though MSCs have several advantages, including self-regeneration ability, antiinflammatory and immunomodulatory effects, and multi-lineage differentiation ability [109,110], keeping them fresh and healthy to maintain their beneficial properties should also be considered.

Optimizing hBM-MSCs While Preserving Cell Quality
As mentioned above, it is imperative to preserve the quality and properties of stem cells, which significantly affect the achievement of many therapeutic cells during ex vivo expansion [56]. Furthermore, before clinical use, MSCs should be isolated and expanded in vitro until they reach the appropriate cell number, owing to the low frequency of 0.001-0.01% of the total mononucleated cells [111]. hBM-MSCs are detached from the culture dish and kept in a largely different environment from the original one, which can diminish their valuable properties [112][113][114]. Hence, maintaining the quality of hBM-MSCs is crucial when using in NDDs. In this section, we summarize several methods of improving the efficiency of BM-MSC therapy while preserving cell stemness.
Donor age is a well-known factor that should be considered during the transplantation process. Many studies have reported that donor age is closely related to negative effects on MSC proliferation and multipotency [115][116][117]. Zaim et al. investigated the effects of donor age and long-term culture on the morphology, characteristics, and capacity of hBM-MSCs to proliferate and differentiate into adipogenic, chondrogenic, osteogenic, and neurogenic lineages [118]. They found that hBM-MSCs gradually reduced their proliferation rate and lost their typical morphology in an age-and passage-dependent manner. In another study, the older donor group showed lower concentrations of colony forming unit fibroblasts (CFU-Fs) and shorter telomere lengths during in vitro expansion than the young donor group, indicating that the ageing of MSCs can reduce their therapeutic characteristics [119].
Moreover, avoiding oxidative stress is effective in maintaining cell freshness. For example, Shin et al. reported that hBM-MSCs trypsinized and maintained in PBS showed a significant loss of freshness and viability over time [57]. This reduction in freshness is accompanied by several phenotypes, such as increased peroxidation of membranes and intracellular vacuoles, in a PBS storage time-dependent manner [58]. In addition, transcriptomic analysis of hBM-MSCs stored in PBS for 12 h predicted increased ROS generation and lipid peroxidation and decreased cell viability [56]. Regarding ROS production, Lee et al. suggested that treatment with N-acetyl-L-cysteine (NAC) and glutathione (GSH) maintained the quality of PBS-stored hBM-MSCs by reducing ROS and lipid peroxidation [56]. Indeed, one study showed that the pre-conditioning of BM-MSCs with NAC led to lower apoptosis and higher survival against oxidative stress by increasing GSH levels and helped bone regeneration after transplantation in rats [120].
Lastly, treatment with growth factors, such as fibroblast growth factor-2 (FGF-2), transforming growth factor-beta (TGF-β), and insulin-like growth factor-1 (IGF-1), also improved the multi-lineage differentiation ability of stem cells [121][122][123]. Nandy et al. reported that FGF-2-treated hBM-MSCs showed the least cell death and the highest upregulation of tyrosine hydroxylase, a dopaminergic neuron marker, compared to other growth-factor-treated cells [121]. Meanwhile, Longobardi et al. reported that the addition of TGF-β and IGF-1 exerted both proliferative and anti-apoptotic actions and even induced the differentiation of BM-MSCs into chondrocytes, showing an increase in the expression of chondrogenic markers in mice [122]. In addition, IGF-1 stimulates osteoblastic differentiation by activating the mTOR signalling pathway and helps maintain bone marrow mass in mice and rats [123]. The effects of substances for improving the efficiency of BM-MSC therapy are summarized in Table 1. Hence, not only the removal of harmful factors such as ROS but also the addition of supplements can be helpful for maintaining hBM-MSC stemness. Table 1. Summary of the functions and effects of substances for improving the efficiency of BM-MSC therapy.

PBS Storage Time Is a Critical Factor for the Differentiation of hBM-MSCs in Gene Expression and Amino Acid Levels
For transplantation, MSCs should be suspended in PBS to prevent inflammatory reactions caused by foreign proteins and serum [124]. To evaluate the effect of storage time on hBM-MSCs before transplantation, transcriptome profiling data of hBM-MSCs stored in PBS for 0, 6, and 12 h were acquired as described in our previous reports [57,58]. The transcriptomic changes in the 6 h-and 12 h-stored groups were compared to the 0 h-stored control groups using Ingenuity Pathway Analysis (IPA) web-based bioinformatics software (Qiagen, CA, USA) and analyzed. Genes that showed >1.5-and <-1.5-fold changes in expression levels were selected and used for transcriptomic analysis. In the 6 h-stored groups, 1466 genes showed such changes in their gene expression levels (806 downregulated and 660 upregulated), whereas in the 12 h-stored groups, 1817 genes showed such changes in their gene expression levels (1145 downregulated and 672 upregulated).
Computational prediction of cellular functions showed that the quantity of ROS, peroxidation of lipid, and cell damage were increased, and that the differentiation of stem cells was inhibited in both 6 h-and 12 h-stored hBM-MSCs compared to the control ( Figure 2). Moreover, these transcriptomic networks showed that ROS levels, lipid peroxidation, cellular damage, and differentiation abilities were related. Interestingly, predictions of all these cellular functions were more robustly represented in the 12 h-storage groups than in the 6 h-storage groups ( Figure 2B), suggesting that ROS production, lipid peroxidation, cell damage, and stem cell differentiation were adversely affected by increasing the PBS storage time in hBM-MSCs. Detailed fold-change information for each gene in the transcriptomic networks is listed in Table 2 (6 h-stored groups) and dishes is inconsistent with in silico prediction and the osteogenic and adipogenic potentials in the previous report [57]. The discrepancies can be attributed to the experimental differences between omics data analyzed directly at each storage time point and long-term cell culture and frequent exchanges of culture medium for cell differentiation. In conclusion, the freshness and differentiation ability of stem cells in stem cell therapy are closely related to the time after cell detachment from culture dishes. In this regard, this bioinformatic study will be an essential factor to be considered in future stem cell therapies.       In the omics analysis approaches, each analysis of single omics has advantages and disadvantages. For example, transcriptomics includes tremendous gene expression data, but it is not able to reflect the exact phenotype of the organism [125]. Metabolomics shows the "end products" of biological processes and reflects more exact phenotypes. However, the metabolites that can be analyzed are limited by experimental methods [126]. Hence, an integrated omics approach can compensate for the shortcomings of each omics and provides a more comprehensive vision of complex biological processes. Indeed, using integrated omics approaches, the unexplained delicate toxicity of nanoparticles and particulate matter [65,[127][128][129] was analyzed, and the biological meaning of mitochondrial diseases was found in various conditions [130].
Amino acid profiling omics data using GC-MS provided cellular information in hBM-MSCs over time [57,131], and the amino acid levels at each time point were integrated with transcriptomics to establish metabotranscriptomics which integrates transcriptomics and metabolomics. Fold changes in the levels of amino acids > 1.2 and <−1.2 were used, and nine and eight amino acids were integrated into the IPA networks of hBM-MSCs stored for 6 h and 12 h, respectively. In detail, in the 6 h-stored groups, GABA (2.289, fold change), glycine (−1.  (2.368) were used for analysis. The metabotranscriptomic networks showed a similar tendency to the transcriptomics prediction, maintaining a more robust prediction of cellular functions in the hBM-MSC group stored for 12 h (Figure 3). These transcriptomic and integrated omics analyses suggest that the PBS storage time is a crucial factor for preserving the cell quality and differentiation ability of hBM-MSCs.
Although our analyses have been reported previously [58], the data in the current review were analyzed using a new, well-developed bioinformatic program. Notably, the data-based program was curated by a data curator from previously published papers, and the results obtained using artificial intelligence are considered more objective biological information rather than data collected by individuals. Our bioinformatic study showing that the freshness of stem cells decreases over time is consistent with previous reports [56][57][58]. However, the differentiation ability of stem cells after cell detachment from culture dishes is inconsistent with in silico prediction and the osteogenic and adipogenic potentials in the previous report [57]. The discrepancies can be attributed to the experimental differences between omics data analyzed directly at each storage time point and long-term cell culture and frequent exchanges of culture medium for cell differentiation. In conclusion, the freshness and differentiation ability of stem cells in stem cell therapy are closely related to the time after cell detachment from culture dishes. In this regard, this bioinformatic study will be an essential factor to be considered in future stem cell therapies.

Conclusions
Herein, we review the application of hBM-MSCs to the treatment of various NDDs and analyze the quality and differentiation ability of hBM-MSCs detached from culture dishes using integrated omics analysis for successful stem cell therapy. Conventional analysis methods have limitations in analyzing the quality and differentiation ability of dissociated hBM-MSCs and cannot comprehensively elucidate the delicate relationships between the cell condition of dissociated hBM-MSCs and therapeutic effects. Multidisciplinary omics and integrated multi-omics approaches provide in-depth and comprehensive information on the quality characteristics and differentiation ability of dissociated hBM-MSCs. Because stemness is a complex process that combines proliferation and self-renewal to generate differentiated cells with identical genotypes, a fragmentary approach is not sufficient to refer to biological changes in the cell. In the future, comprehensive and computational analyses of dissociated hBM-MSCs at the genomic, transcriptomic, small RNAomic, proteomic, phenomic, and metabolomic levels using advanced machine learning algorithms will accelerate studies in the field of stem cell therapy. Thus, these

Conclusions
Herein, we review the application of hBM-MSCs to the treatment of various NDDs and analyze the quality and differentiation ability of hBM-MSCs detached from culture dishes using integrated omics analysis for successful stem cell therapy. Conventional analysis methods have limitations in analyzing the quality and differentiation ability of dissociated hBM-MSCs and cannot comprehensively elucidate the delicate relationships between the cell condition of dissociated hBM-MSCs and therapeutic effects. Multidisciplinary omics and integrated multi-omics approaches provide in-depth and comprehensive information on the quality characteristics and differentiation ability of dissociated hBM-MSCs. Because stemness is a complex process that combines proliferation and self-renewal to generate differentiated cells with identical genotypes, a fragmentary approach is not sufficient to refer to biological changes in the cell. In the future, comprehensive and computational analyses of dissociated hBM-MSCs at the genomic, transcriptomic, small RNAomic, proteomic, phenomic, and metabolomic levels using advanced machine learning algorithms will accelerate studies in the field of stem cell therapy. Thus, these approaches will be helpful in analyzing the condition of dissociated hBM-MSCs and improving their quality and differentiation ability for innovative and successful stem cell therapy.