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. 2023 Dec 7;32:09636897231213271. doi: 10.1177/09636897231213271

Preclinical Short-term and Long-term Safety of Human Bone Marrow Mesenchymal Stem Cells

Ziyang Liang 1,2,*, Guoyang Zhang 1,2,*, GuangTing Gan 1,2,3,*, Duolan Naren 1,4, Xiaoyan Liu 1,2, Hongyun Liu 1,2, Jiani Mo 1,2, Shengqin Lu 1,2, Danian Nie 1,2, Liping Ma 1,2,✉
PMCID: PMC10704945  PMID: 38059278

Abstract

Mesenchymal stem cells (MSCs) have become a promising therapeutic method. More safety data are needed to support clinical studies in more diseases. The aim of this study was to investigate the short- and long-term safety of human bone marrow–derived MSCs (hBMMSCs) in mice. In the present study, we injected control (saline infusion only), low (1.0 × 106/kg), medium (1.0 × 107/kg), and high (1.0 × 108/kg) concentrations of hBMMSCs into BALB/c mice. The safety of the treatment was evaluated by observing changes in the general condition, hematology, biochemical indices, pathology of vital organs, lymphocyte subsets, and immune factor levels on days 14 and 150. In the short-term toxicity test, no significant abnormalities were observed in the hematological and biochemical parameters between the groups injected with hBMMSCs, and no significant damage was observed in the major organs, such as the liver and lung. In addition, no significant differences were observed in the toxicity-related parameters among the groups in the long-term toxicity test. Our study also demonstrates that mice infused with different doses of hBMMSCs do not show abnormal immune responses in either short-term or long-term experiments. We confirmed that hBMMSCs are safe through a 150-day study, demonstrating that this is a safe and promising therapy and offering preliminary safety evidence to promote future clinical applications of hBMMSCs in different diseases.

Keywords: bone marrow–derived mesenchymal stem cells, long-term safety, preclinical safety

Introduction

In the last decade, mesenchymal stem cells (MSCs) have been widely used to repair organ damage and inflammatory diseases due to their lack of expression of costimulatory molecules and low immunogenicity, as well as their strong differentiation capacity, immunomodulatory ability, and low toxicity effects1–5. By searching the Clinical Trials data, we can see that there are 1,273 stem cell–related clinical trials retrieved worldwide (source: http://www.clinicaltrials.gov, “Mesenchymal stem or Mesenchymal Stromal,” queried in December 2022), which are mainly used in various diseases, such as respiratory, endocrine, neurological, motor, immune, reproductive, circulatory, and digestive system diseases. More than 20 stem cell drugs have been approved for marketing internationally, the majority of which belong to bone marrow mesenchymal stem cells (BMMSCs) for indications including graft-versus-host disease (GVHD), Crohn’s disease, acute myocardial infarction, myasthenia gravis, severe lower limb ischemia, and Alzheimer’s disease6,7. Even in Europe and Japan, the use of MSCs has been approved in GVHD and Crohn’s disease 8 . This progress underscores the tremendous therapeutic potential of MSCs.

Since the original isolation of BMMSCs from bone marrow by Friedenstein et al. 9 , they have been extensively used in the treatment of various diseases due to their high abundance in the bone marrow, their strongest differentiation capacity, and the ease of extraction, which can be obtained by simple bone marrow puncture10,11. It is the only one among all types of stem cells that can promote the maturation of hematopoietic stem cells (HSCs) in vivo 12 . The role of immunomodulatory mechanisms of BMMSCs is primarily achieved through direct intercellular contacts or indirect paracrine secretion. The paracrine effect is currently understood as the secretion of soluble mediators such as transforming growth factor-β (TGF-β), Prostaglandin E2 (PGE2), interleukin (IL)-6, IL-10, and Indoleamine 2,3-dioxygenase (IDO). These mediators act on dendritic cells, monocytes, natural killer cells, and other innate immune cells, influencing their effector functions and phenotypes13–15. We also found that mouse-derived BMMSCs rapidly increased platelet counts, promoted the secretion of inflammatory factors (IL-10, TGF-β), and upregulated Treg cell ratios in Immune thrombocytopenia (ITP) mice, thereby improving immune disorders 16 . In recent years, to overcome the disadvantages of long culture cycles in vitro and the tendency to lose the physiological properties of MSCs, bone marrow–derived clonal mesenchymal stem cells (BM-cMSCs) have offered a potential method to produce homogeneous MSCs 17 . The establishment of an allogeneic bone marrow–mesenchymal stem cell bank from different populations by means of sub-differentiated culture of bone marrow samples from healthy and young volunteers can overcome the disadvantages of low productivity and high heterogeneity 18 . In conclusion, these studies suggest that BMMSCs may be a promising candidate for cell-based therapies for autoimmune and inflammatory diseases.

Safety concerns regarding the use of MSC therapy appear to be a major constraint in the development of MSC-based treatments for a wide range of human diseases 19 , which is also the reason why it is difficult to carry out clinical studies on MSCs for some diseases. In addition to in vitro studies, reliable evaluation in preclinical studies is required to confirm the safety and nontoxicity of the product 20 . Preclinical evaluations are necessary to develop clinical-grade BM-cMSCs as an advanced therapeutic agent 21 . Here, preclinical studies of human bone marrow–derived MSCs were conducted to evaluate the safety of hBMMSCs in animal models, and these findings will be used to advance clinical studies on MSCs.

Materials and Methods

Cell Preparation

Extraction and culture of hBMMSCs were performed by Guangzhou Saliai Stem Cell Science and Technology Co., LTD. The hBMMSCs were tested by the China Academy of Food and Drug Administration and conformed to the Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations.

In addition, the adipogenic and osteogenic differentiation ability must be positive, and the tests for bacteria, fungi, mycoplasma, hepatitis virus, and endotoxin must all be negative. Insulin, isobutylmethylxanthine, indomethacin, and dexamethasone were added to the basal medium of MSCs to induce adipogenesis. After day 21, adipocytes were stained with Oil Red O (Millipore, Billerica, MA, USA). Ascorbic acid, dexamethasone, and β-glycerol phosphate were added to the culture to induce osteogenesis. Differentiated cells were stained with alizarin red on day 21 or 28 to detect osteogenic mineralization.

Immunophenotypes were identified according to the International Society for Cellular Therapy standards, which defines human MSCs as expressing CD105, CD90, and CD73 but lacking CD45, CD34, CD11b, CD19, and HLA-DR expression (Fig. 1) 22 . For flow cytometry analysis, cells were stained with human anti-CD45, CD34, CD11b, CD19, HLA-DR, CD105, CD73, and CD90 antibodies (BD Pharmaingen, USA) conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Studies were conducted using P4 generation cells for preclinical trials.

Figure 1.

Figure 1.

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Characterization of hBMMSCs. (A and B) Fourth-passage BMMSCs display fibroblast-like spindle morphology, 200× and 400×, respectively. (C and D) BMMSCs show the potential to differentiate into adipose (Oil Red staining), osteogenic (alizarin red) lineages. (E) BMMSCs were negative for CD11b, CD19, CD45, HLA-DR-A, and CD34, but positive for CD105, CD73, and CD90. (F) BMMSCs were analyzed using standard G-banding karyotyping, which identified a typically normal diploid male chromosomal constitution. Karyotype analysis of five mitotic cells showed no abnormalities. hBMMSCs: human bone marrow–derived mesenchymal stem cells; BMMSCs: bone marrow mesenchymal stem cells.

Fifth-generation karyotyping of hBMMSCs was performed using standard high-resolution G-banding protocols. hBMMSCs were thawed in a water bath at 37°C, centrifuged, resuspended in appropriate amounts of saline, and injected into the animals.

Mice

The experimental procedures were conducted under ECDirective 86/609/EEC for animal experiments, and our experiments comply with current valid legal and ethical recommendations in China. It was also approved by the Sun Yat-Sen University Medical Center Ethics Committee (No. SYSU-IACUC-2022-001942). Both toxicity experiments and abnormal immune response tests were performed using BALB/c mice. Female BALB/c mice (n = 24, 18–20 g; 8–10 weeks old) and male BALB/c mice (n = 24, 18–20 g; 8–10 weeks old) were purchased from Charles River Laboratories, China.

Short-term Toxicity Tests

Twenty-four BALB/c mice were divided into four groups (vehicle control, low-dose, medium-dose, and high-dose groups, with three females and three males in each group) for 2-week short-term toxicity experiments. The vehicle control group was injected with 200 µl of 0.9% NaCl solution in the tail vein, while the low-, medium-, and high-dose groups were injected with 1.0 × 106/kg, 1.0 × 107/kg (a 10-fold dose relative to patients), and 1.0 × 108/kg (a 100-fold dose relative to patients) hBMMSCs (dissolved in 200 µl of saline), respectively. The day of hBMMSC injection was defined as day 1 (D1), mice were sacrificed at D14, and necropsy and gross pathological examinations were performed. With intraperitoneal injection of barbiturates, the mice were killed by dislocating the cervical vertebrae and severing the spinal cord after complete anesthesia, and it was confirmed that the mice’s heartbeat had completely stopped and pupils were dilated. Body weight, body temperature, and clinical sign scores 23 (Table 1) of the mice were measured every 3 days starting from D0. Hematological and biochemical analyses were performed at D0, D1, D3, D7, and D14 by blood collection from the medial canthus vein (final volume 150 µl) to compare the baseline status and the changes after stem cell injection. All blood samples were analyzed by an automatic blood cell analyzer (BC-2800vet, Mindray, ShenZhen, China). The levels of albumin, aspartate transaminase (AST), alanine aminotransferase (ALT), serum creatinine (Scr), and blood urea nitrogen (BUN) were measured at each time point using a biochemical assay kit (Elabscience, China) separately.

Table 1.

Clinical Sign Scores.

Parameter Points
Body weight
 Unaffected 0
 Variation <5% 1
 Weight reduction 5%–10% 5
 Weight reduction 11%–20% 10
 Weight reduction >20% 20
General state
 Fur smooth and shiny, stoma clean, eyes clear and shiny 0
 Fur faulty (reduced/excessive body hygiene) 1
 Fur lusterless, disheveled, scruffy stoma, eyes turbid, increased muscle tone 5
 Dirty fur, sticky or moist stoma, abnormal posture, eyes turbid, increased muscle tone 10
 Cramps, paralysis (trunk muscle, limbs), breath sounds, cold temperature 20
Spontaneous behavior
 Normal behavior (e.g., sleep, reaction to blow and touch, curiosity, social contacts) 0
 Slight variation to normal behavior (e.g., reduced curiosity, reduced exploratory behavior) 1
 Unusual behavior (e.g., limited motor function, hyperkinetic, reduced mobility) 5
 Self-isolating, lethargy, distinct hyperkinetic, stereotypy of behavior, coordination disorder 10
 Pain noises by grabbing, self-amputation (automutilation) 20
Treatment specific
 Neurotoxicity (stagger, loss of sensitivity) 10
 Anemia (pale/cyanotic skin) 10
 Superficial bleeding/hematoma 10
 Enlarged spleen/lymph nodes 20
 Soft stool 5
 Persistent diarrhea 20
 Heavy infection (high body temperature, purulent secretory discharge) 20
 Acute kidney failure (edema, uremia, lethargy, hyperventilation) 20
Degree of strain (DS)
 DS0 = no strain 0
 DS1 = minor strain 1–9
 DS2 = moderate strain 10–20
 DS3 = severe strain >20
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Venous blood and spleen samples were collected from mice after 1 h and 1 day of infusion of BMMSCs, respectively. The peripheral blood cells and spleen monocytes were incubated with anti-human CD45-APC and CD73-FITC antibodies (Biolegend, USA) for 30 min at 4°C in the dark.

Critical organ (heart, liver, lungs, spleen, lymph nodes, bone marrow, kidneys, genitals) histology was evaluated with formalin-fixed, hematoxylin and eosin (H&E)-stained sections in a blinded manner. Acute and chronic inflammatory responses, coagulative necrosis, fatty changes, hemorrhage, congestion, and any changes in normal tissue structure were assessed in different samples.

Long-term Toxicity Tests

In the long-term toxicity tests, they were also divided into four groups with the same dose as described above for a 150-day test: vehicle control, low-dose, medium-dose, and high-dose groups (hBMMSCs were injected at the same quantity as in the short-term toxicity experiments). Blood was collected through the medial canthus vein 150 days after tail vein injection in four groups of mice for relevant tests, and relevant organs were retained for pathological examination. The following indices were measured: body weight (once per month), body temperature (once per month), hematological (D150), biochemical analysis (D150), and necropsy and gross pathological examinations (D150).

Abnormal Immune Response Test

This study conducted 1-, 3-, 14-, and 150-day tests to examine whether MSCs induce any abnormal immune abnormalities in the short or long term (grouping and use of mouse species as in toxicity experiments). In a 150-day experimental test, blood was extracted from mice at D0, D1, D3, D14, and D150, and the immune response to hBMMSCs was detected using antibodies against the cell surface markers CD3-PE or CD3-FITC, CD4-FITC or CD4-APC, and CD8a-PerCP or CD8a-PE (BD Pharmaingen, USA) to analyze immune cell subpopulations in the blood of each group (grouped as before) of mice after intravenous injection, and isotype control was performed by flow cytometry. Serum levels of interleukin-4 (IL-4), interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-17 (IL-17), interferon-gamma (IFN-γ), and transforming growth factor-β (TGF-β) were also measured using enzyme-linked immunosorbent assay (ELISA) kits (Mlbio, China or Elabscience, China).

Statistical Analysis

All results are expressed as the mean ± standard deviation. The obtained data were analyzed using SPSS 22.0 statistical software. Comparisons between groups were performed by analysis of variance (ANOVA) followed by the Dunnett t-test when results from ANOVA were significant. P < 0.05 was considered to be statistically significant.

Results

Cell Characterization

BMMSCs displayed a fibroblast-like or spindle-shaped morphology, with approximately 80% to 90% fusion (Fig. 1A, B). Immunophenotypic analysis of the isolated BMMSCs revealed high expression levels of MSC markers (CD73, CD90, and CD105), while hematopoietic markers (CD19, CD34, CD45, CD11b, HLA-DR) were either low or absent (Fig. 1E). The pluripotency of the isolated MSCs was confirmed through their ability to differentiate into lipogenic and osteogenic lineages in vitro (Fig. 1C, D). In addition, standard g-band karyotyping demonstrated that the isolated BMMSCs, which had undergone four passages, exhibited normal diploid male karyotypes without any observed karyotypic abnormalities (Fig. 1F).

hBMMSCs Have No Adverse Effects in Short-term Toxicity Tests in Mice

In the 14-day short-term safety tests, no mortality or abnormal clinical signs were observed in any of the four groups of mice. According to the results, there was a slight decrease in body weight of the four groups of mice on the third day after treatment, but the body weight of all mice gradually increased in subsequent observations, in accordance with the growth pattern of mice. The reason for the weight loss on the third day was probably related to the blood extraction and the weight loss that occurred in the control group, but it was not related to the injection of hBMMSCs. The total body weight of mice was not statistically significant compared with the control group in each of the 3 days of body weight testing (Fig. 3A). In addition, none of the mice developed fever, and the body temperatures of the three treatment groups were not significantly different from those of the control group (Fig. 3A). Regarding clinical symptom scores, all four groups of mice showed a score of 0 for subsequent clinical symptoms, except for the third day when the score increased due to weight loss (Fig. 3A).

Figure 3.

Figure 3.

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Hematological and biochemical parameters of mice in the short-term toxicity study of human bone marrow–derived mesenchymal stromal cells on day 14. (A) Changes in body weight, body temperature, and clinical symptom scores in mice during a 14-day short-term toxicity study. (B) Influence of BMMSCs infusion on blood cell parameters in mice, including red blood cell count, hemoglobin, HCT, platelets, leukocytes, neutrophils, platelets, lymphocyte ratio, and monocyte ratio. (C) Effect of BMMSCs on biochemical indices in mice. Values are represented as the mean ± SD, *P ≤ 0.05 compared with the vehicle control, *P < 0.05; indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 6 mice/group. BMMSCs: bone marrow mesenchymal stem cells; HCT: hematocrit; RBC: red blood cell; WBC: white blood cell; HGB: hemoglobin; NEUT: neutrophil; PLT: platelet; ALB: albumin; ALT: alanine aminotransferase; AST: aspartate transaminase; BUN: blood urea nitrogen.

Most of the hematological and biochemical evaluations showed no significant differences in any laboratory parameters between the control and treatment groups (Figs. 2C, D and 3B, C). A mild decrease in white blood cell (WBC) count of 1.0 × 108/kg hBMMSCs was observed on day 3 but was not statistically significant compared with the control group (P = 0.0566) and recovered quickly on day 7 (Figs. 2C and 3B). Similarly, in the high-dose group, a decrease in erythrocytes was observed on the seventh day of infusion compared with the control group (P < 0.05). However, all these values returned to normal on the 14th day (Fig. 3B). In addition to experiments on the distribution of MSCs in blood and bone marrow, we collected peripheral blood 1 h after the infusion of stem cells and bone marrow 1 day after the infusion. The results revealed the presence of infused hBMMSCs in both blood and bone marrow, confirming the absence of mis-injection of MSCs (Fig. 2A, B). Furthermore, the level of hBMMSCs in the mice bone marrow was significantly higher in the high-dose group compared with the other three groups. This finding may explain the temporary decrease in red blood cell (RBC) and WBC, as the homing of MSCs resulted in a transient decline of blood cells.

Figure 2.

Figure 2.

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The distribution of BMMSCs in each group of mice after infusion, as well as the changes in hematological and biochemical indices during the first 3 days. (A) Blood samples were collected from each group of mice 1 h after the infusion of BMMSCs. After 24 h, bone marrow monocytes were collected. The presence of hBMMSCs in blood and bone marrow was examined using flow cytometry. The hBMMSCs were stained with anti-human CD45 and CD73 flow antibodies. The CD45-CD73+ cells represented h-BMMSCs. (B) After infusion of BMMSCs, they were detected in both blood and bone marrow in dose-dependent amounts. (C) Influence of BMMSCs infusion on blood cell parameters in mice, including red blood cell count, hemoglobin, HCT, platelets, leukocytes, neutrophils, platelets, lymphocyte ratio, and monocyte ratio. (D) Effect of BMMSCs on biochemical indices in mice. Values are represented as the mean ± SD; nsP > 0.05, **P ≤ 0.05, ****P ≤ 0.0001, P = 0.56: high-dose versus control; Indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 6 mice/group. ns: no statistical significance. BMMSCs: bone marrow mesenchymal stem cells; hBMMSCs: human bone marrow–derived mesenchymal stem cells; HCT: hematocrit; APC: allophycocyanin; FITC: fluorescein isothiocyanate; RBC: red blood cell; HGB: hemoglobin; NEUT: neutrophil; PLT: platelet; WBC: white blood cell; ALB: albumin; ALT: alanine aminotransferase; AST: aspartate transaminase; BUN: blood urea nitrogen.

Figure 4 shows representative images of the H&E-stained heart, liver, kidney, lung, spleen, lymph nodes, bone marrow, and gonad tissues and the corresponding organ weights. Histopathological evaluations did not show alterations such as necrosis or inflammatory reactions in the treated groups (Fig. 4B). Overall, all collected tissues were normal, and histopathological examination excluded the occurrence of any damage and toxicity. In addition, there was no statistical significance (P > 0.05) for each organ in the three treatment groups compared with the control group, both in terms of weight and percentage of organ to body weight (Fig. 4A).

Figure 4.

Figure 4.

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Weight and HE staining results in important organs of mice injected with BMMSCs for 2 weeks. (A) Final organ weights or organ-to-body weight ratios for male and female mice injected with BMMSCs for the 2-week short-term toxicity study. (B) H&E staining of parts of important organs of mice. Magnification, ×400; scale bar, 50 μm. Values are represented as the mean ± SD; indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 6 mice/group. HE: hematoxylin and eosin; BMMSCs: bone marrow mesenchymal stem cells.

hBMMSCs Have No Adverse Effects in Long-term Toxicity Tests in Mice

Most of the current safety studies have focused on short-term responses, but MSCs, unlike drugs, can be continuously reproduced in vivo, so long-term toxicity testing is essential. Therefore, hematological and histopathological examinations were performed 150 days after hBMMSC infusion. In long-term toxicity tests, the weight (P ≥ 0.05) and body temperature (P ≥ 0.05) of mice treated with the different doses of hBMMSCs were not statistically significant compared with the control group and were in the normal range (Fig. 5A). For the hematological parameters, all the data (e.g., hemoglobin, WBC, platelet (PLT) of the four groups were in the normal range, and no difference was found between the treatment groups and the control group (Fig. 5B). Importantly, H&E staining showed no difference in vital viscera between mice with pretreated hBMMSCs and control mice (Fig. 5C). Similarly, their organ weight comparisons were not statistically significant. Thus, hBMMSCs appear to be safe in the long or short term.

Figure 5.

Figure 5.

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BMMSCs do not influence long-term toxicity in mice. (A) Changes in body weight and body temperature of mice 150 days after BMMSCs injection. (B) Variety of blood indices, including RBC, HGB, and HCT, and the percentage (%) of blood cells, including WBC, PLT, and LYM, in mice 150 days after BMMSCs injection. (C) Weight, HE staining results in important organs of mice injected with BMMSCs for 150 days. Magnification, ×400; scale bar, 50 μm. Values are represented as the mean ± SD; indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 4 mice/group. BMMSCs: bone marrow mesenchymal stem cells; RBC: red blood cell; HCT: hematocrit; WBC: white blood cell; HGB: hemoglobin; LYM: lymphocyte; PLT: platelet; HE: hematoxylin and eosin.

Infusion of hBMMSCs Does Not Elicit Abnormal Immune Responses

To assess whether hBMMSCs cause abnormal immune responses in vivo, we used flow cytometry and ELISA to detect relevant immune indicators, not only for short-term observations but also for long-term (150 day) observations. No significant differences were observed in the percentages of CD3+, CD4+, and CD8+ T lymphocytes in mice treated with hBMMSCs compared with the control on D1, D3, and D14. In addition, serum levels of IL-17, IL-4, IL-2, IL-10, IFN-γ, and TGF-β were also found to be similar between the vehicle and cell treatment groups (Fig. 6). Similar results were obtained 150 days after the infusion of hBMMSCs, and no significant changes were observed in the lymphatic subpopulations or in the levels of inflammatory factors in the mice (Fig. 7). These data suggest that infusion of hBMMSCs did not show any measurable short- or long-term effects on abnormal immunity.

Figure 6.

Figure 6.

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Variation in lymphocyte subpopulations and cytokine levels in the serum samples of mice on the first 3 days and the 14th day after BMMSCs injection. (A, B) The results showed that there were no significant differences in CD4+ and CD8+ ratios, as well as CD4+/CD8+ levels, between the four groups during the first 3 days and on day 14. (B) No significant differences in the levels of cytokines were observed between cell- and vehicle-treated animals. Values are represented as the mean ± SD; indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 6 mice/group. BMMSCs: bone marrow mesenchymal stem cells; IFN-γ: interferon-gamma; IL: interleukin; TGF-β: transforming growth factor-β.

Figure 7.

Figure 7.

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Variation in lymphocyte subpopulations and cytokine levels in the serum samples of mice 150 days after BMMSCs injection. (A) After 150 days of injection of BMMSCs, no significant differences in CD4+ or CD8+ ratios or CD4+/CD8+ levels were observed among the four groups. (B) No significant differences in the levels of cytokines were observed between cell- and vehicle-treated animals. Values are represented as the mean ± SD; indicators not mentioned in the figure were not statistically significant when compared with the control group. The corresponding P values and confidence intervals are provided in Supplementary Table 1; N ≥ 4 mice/group. BMMSCs: bone marrow mesenchymal stem cells; FITC: fluorescein isothiocyanate; IL: interleukin; INF-γ: interferon-γ; TGF-β: transforming growth factor-β.

Discussion

MSCs have been widely used in the treatment of various diseases for the past 20 years due to their excellent differentiation ability and immunomodulatory capacity. In contrast, BMMSCs, the first isolated mesenchymal stem cells, have been shown to have good therapeutic effects in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and GVHD24,25. Particularly, in the treatment of IBD, BMMSCs have demonstrated clear efficacy. Two phase I studies have shown clear efficacy in both systemic administration and local injection therapy 26 . Moreover, we demonstrated that BMMSCs significantly elevated platelet counts in ITP mice and could enhance Treg cell ratios and reverse immune disorders in ITP mice 16 . In contrast to other MSCs, BMMSCs are not only clinically accessible and have similar immunomodulatory capacity as adipose-derived MSCs27,28, but more importantly, they are the only MSCs that have been shown to promote HSC maturation and expansion in an in vivo test in mice 12 . Therefore, we would like to select a single-donor in vitro clone of BMMSCs for further ITP clinical trials. This would significantly reduce not only the amounts of cells used but also in terms of efficacy. However, it is crucial to consider the potential genetic alterations that may occur during extended in vitro culture, as these alterations could affect the safety of MSCs when used in vivo. Therefore, conducting preclinical safety studies on hMSC biologics before initiating clinical trials is essential. These studies can help identify and predict any potential toxic reactions or immune abnormalities that may arise from the use of biologics. In recent years, several preclinical studies have investigated the safety of hMSCs, most of them focused on topical treatments such as intrathecal injections and ocular administration29–31. A recently published clinical trial in the Netherlands showed promising results with intranasal injection of BMMSCs in patients with perinatal arterial ischemic stroke 32 , and no serious adverse effects were observed in patients followed up to 3 months of age. It is important to note that most of the aforementioned studies focused on localized drug administration, and there is currently fewer available safety evaluation for systemic injection of human mesenchymal stromal cells (HMSCs). It was once expected that MSCs could survive in vivo for a long time after infusion, similar to HSCs. However, extensive studies have shown that infused MSCs do not survive for long periods of time in the living body. When MSCs were infused into both humans and animals, a large amount of MSCs was observed to accumulate in the lungs, and most of the MSCs were cleared in the following 3 to 6 days. After 1 week, less than 1% of MSCs were distributed in various organs of the body, including the spleen, liver, and bone marrow 33 . However, this does not mean that MSCs are not toxic in the long term because monocytes performing clearance functions produce phenotypic changes after phagocytosis of exogenous MSCs, producing a large number of inflammatory factors and inducing Treg production. There is a long-term effect on the immune environment in the body 34 . Therefore, long-term toxicity and abnormal immunity tests are essential. Therefore, we designed three escalating levels of hBMMSCs according to the clinical trial design route of delivery and dose of administration and then assessed their safety in mice in terms of short- and long-term toxicity and abnormal immune responses.

In the short- and long-term toxicity tests, we examined the general condition, blood tests, liver and kidney functions, and pathological changes of each organ in four groups of mice injected with different concentrations of gradient cells at different time periods. As mentioned, these items are essential variables for assessing the standard toxicity and safety of drugs and biological agents. Short- and long-term systemic injections of hBMMSCs did not result in significant systemic toxicity at various clinical and preclinical levels compared to control female and male mice. However, there was a temporary decrease in WBC and RBC levels in the early stage of infusion, which recovered within a short period of time. This decrease may be attributed to the homing of BMMSCs to the bone marrow. In order to avoid any potential impact on the function of MSCs, we refrained from using in vitro stains to label MSCs 35 . Instead, we directly detected the labeling of BMMSCs in the mouse bone marrow using flow cytometry. After 1 day, we observed a significantly higher presence of hBMMSCs in the bone marrow of the high-dose group compared with other groups. This observation may explain the transient decrease in WBCs and RBC during the early stage of the high-dose group, which could be associated with the impact on hematopoiesis. Although a risk of pulmonary embolism has been reported after the infusion of adipose-derived MSCs 36 , especially after injection concentrations above 1.0 × 107 cells/ml 37 , we used 200 µl of physiological saline to dilute the cells and infused them into the mice at a slow rate. Therefore, even after the infusion of high doses of cells, the mice did not suffer from respiratory distress or even sudden death. In addition, pathological histopathological examination did not show necrosis, inflammatory reactions, or other changes in the three test groups. Similar results were obtained by Tayebi et al. 38 They also used hBMMSCs in acute and chronic toxicity tests that were also performed and evaluated by detecting behavioral changes, clinical signs of toxicity, changes in body weight, water and food intake, and histopathology of target tissues. Both males and females were included in the study, but they were only evaluated at a single dose, and there was no concentration creep, which is clearly not sufficient in safety testing. In another earlier study on the safety of hBMMSCs 39 , although a concentration gradient was also performed, it only examined the general status after 30 days of infusion, including weight and clinical symptoms, without involving tests such as blood biochemistry.

MSCs uniquely exhibit low immunogenicity, lack major histocompatibility complex (MHC)-II expression, express low levels of MHC-I, and are not altered by lymphocytes 40 , which reduces their chances of eliciting an immune response at transplantation. However, the abnormal immune response caused by allogeneic cell infusion cannot be ignored, especially when we apply it to the treatment of autoimmune diseases. We investigated the immune status of mice before and after cell injection, focusing on subpopulations of lymphocytes in peripheral blood and six major inflammatory factors. The findings revealed that there were no significant changes in the immune environment of the mice during both the early and late infusion periods. Moreover, the infusion of hBMMSCs did not lead to any abnormal immune disorders compared to the control group. Similar conclusions were observed by Rengasamy et al. 41 They investigated abnormal immune responses at 30 and 90 days after administering hBMMSCs. However, they only detected three proinflammatory factors. In our study, we conducted a comprehensive examination of the changes in the immune environment of mice injected with hBMMSCs. We extended the observation period and analyzed a wider range of immune indicators. Our findings demonstrated that hBMMSCs did not elicit any abnormal immune responses.

In conclusion, the safety assessment conducted in this study suggests that intravenous administration of hBMMSCs is safe, as no severe adverse effects were observed even after 150 days. These findings provide evidence to support the potential use of hBMMSCs in various diseases in future clinical applications.

Supplemental Material

sj-docx-1-cll-10.1177_09636897231213271 – Supplemental material for Preclinical Short-term and Long-term Safety of Human Bone Marrow Mesenchymal Stem Cells
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Supplemental material, sj-docx-1-cll-10.1177_09636897231213271 for Preclinical Short-term and Long-term Safety of Human Bone Marrow Mesenchymal Stem Cells by Ziyang Liang, Guoyang Zhang, GuangTing Gan, Duolan Naren, Xiaoyan Liu, Hongyun Liu, Jiani Mo, Shengqin Lu, Danian Nie and Liping Ma in Cell Transplantation

Acknowledgments

We thank the Lim Por Yen Medical Research Center of Sun Yat-Sen Memorial Hospital (Guangdong, China) for their equipment support and technical guidance, and also thank Saliai Stem Cell Science and Technology Co. LTD for providing hBMSCs.

Footnotes

Author Contributions: L.M. and Z.L. designed the research and conceived of the manuscript; Z.L., G.Z., and G.G. implemented the experimental studies and wrote the paper together. They have contributed equally to this work and share first authorship. X.L., H.L., J.M., and S.L. were responsible for specimen collection. G.Z. and L.M. modified the draft of the paper; all authors approved the final version of manuscript.

Availability of Data and Materials: The data are available from the corresponding author upon reasonable request.

Ethical Approval: This study protocol was reviewed and approved by the Sun Yat-Sen University Medical Center Ethics Committee, approval number SYSU-IACUC-2022-001942.

Statement of Human and Animal Rights: All procedures in this study were conducted in accordance with the Sun Yat-Sen University Medical Center Ethics Committee’s approved protocols (approval number SYSU-IACUC-2022-001942).

Statement of Informed Consent: There are no human subjects in this article and informed consent is not applicable.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Science and Technology Program of Guangzhou (grant numbers 201803010012; 202201010936).

Supplemental Material: Supplemental material for this article is available online.

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Supplementary Materials

sj-docx-1-cll-10.1177_09636897231213271 – Supplemental material for Preclinical Short-term and Long-term Safety of Human Bone Marrow Mesenchymal Stem Cells
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Supplemental material, sj-docx-1-cll-10.1177_09636897231213271 for Preclinical Short-term and Long-term Safety of Human Bone Marrow Mesenchymal Stem Cells by Ziyang Liang, Guoyang Zhang, GuangTing Gan, Duolan Naren, Xiaoyan Liu, Hongyun Liu, Jiani Mo, Shengqin Lu, Danian Nie and Liping Ma in Cell Transplantation

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