Bio-distribution and toxicity potential of human umbilical cord mesenchymal stem cells in cynomolgus monkeys

Mesenchymal stem cells (MSCs) have demonstrated promising advantages in the therapies of many diseases, while its multi-directional differentiation potential and immunotoxicity are the major concerns hindered their clinical translation. In this study, human umbilical Mesenchymal stem cell (hUC-MSCs) were labeled with a near-infrared fluorescent dye DiR before infused into cynomolgus monkeys, and the amount of hUC-MSCs in the peripheral blood were dynamically estimated from 5 min to 28 days post a single administration at 3 × 106 cells/kg and 2 × 107 cells/kg intravenously. As results, some hUC-MSCs distributed to the whole body within 5 min, while most of the cells accumulate in the lungs along with the systemic blood circulation, and subsequently released into the blood. The toxicity potentials of hUC-MSCs were investigated in another 30 cynomolgus monkeys, and the cells were repeatedly administrated at doses of 3 × 106 cells/kg and 2 × 107 cells/kg for 5 times on a weekly basis, with a recovery period of 1 months. hUC-MSCs showed no obvious toxic effects in cynomolgus monkeys, except xenogeneic immune rejection to human stem cells. Low levels of the hUC-MSC gene were detected in the peripheral blood of a few animals administered 2 × 107 cells/kg at 30 min subsequent to the first and last administration, and there was no significant difference in the copy number of the hUC-MSC gene in the blood samples compared with the first and last administration, indicating that the hUC-MSC was not significantly amplified in vivo, and it its safe in non-human primates. Our study for the first time verified the safety of long-term use of hUC-MSCs in primates. We have pioneered a technology for the real-time detection of hUC-MSCs in peripheral blood and provide dynamicand rapid monitoring of the distribution characteristics of hUC-MSCs in vivo. Here, we provide data supporting the application of such products for clinical treatment and the application of stem cells in major refractory diseases and regenerative medicine.


Cells
hUC-MSCs at GMP-grade quality were provided by Sinoneural Cell Engineering Group Co., Ltd. and stored in liquid nitrogen away from light.The stem cells were recovered and using solvent (saline containing 5% DMSO and 5% human blood albumin, the DMSO was further diluted and the final concentration in the cell suspension was 0.25%), and were adjusted to a certain concentration before use, and the viability was estimated as 89.00% ± 3.43%.

Animals
A total 34 of cynomolgus monkey (Macaca fascicularis) aged 3-4 years were purchased from Guangdong Blue Island Biotechnology Co., Ltd.(Experimental animal production license number: SCXK [Yue] 2019-0010).The the body masses of females ranged from 2.50 to 3.25 kg, and the body masses of males ranged from 2.55 to 3.80 kg.The temperature of the rearing room was 16-26 °C, the relative humidity was 40-70%, and the lighting time was 12 h/day.All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Center for Safety Evaluation of Drugs (No. IACUC-2020-069).The study is presented in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Groups and administration
There are four animals used in the bio-distribution study with DiR, and 30 animals in the toxicity study.All the cynomolgus monkey for the toxicity study were randomlised into 3 groups, including Control Group, Low-dose Group and High-dose Group, with 5 males and 5 females in each.These animals were administered intravenously (intravenous infusion at 3 mL/min) with solvent or hUC-MSCs at 3 × 10 6 cells/kg or 2 × 10 7 cells/kg at 10 mL/ kg for once a week for 4 consecutive weeks (five times in total), and a 4-week recovery period was set after the last administration.
The intended clinical dose of hUC-MSCs is 1 × 10 6 cells/kg.In terms of kilogram body weight, the low dose (3 × 10 6 cells/kg) and high dose (2 × 10 7 cells/kg) used in this study was 3 times and 20 times the clinical equivalent dose, respectively.

Bio-distribution of hUC-MSCs with DiR
hUC-MSCs were rinsed with PBS for twice before use.Dye of 1, 1′-dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DiR, Caliper Life Sciences) was diluted to 5 μg/mL andmixed with the hUC-MSCs (for 1 × 10 6 cells, 1 mL of DiR dye was required).The mixture was incubated in a 37 °C incubator for 30 min, and the supernatant was removed by centrifugation at 400 g for 5 min.The cells were rinsed again in PBS for twice by centrifugation.Four monkeys were used in this study.DiR-labeled hUC-MSCs were administered to monkeys by a single intravenous injection at doses of 3 × 10 6 cells/kg or 2 × 10 7 cells/kg respectively.Peripheral blood of 500 μL was collected at before, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h and 3 h, 1, 2, 3, 5, 7, 10, 14, 21, and 28 days post-administration to determine the distribution of hUC-MSCs in blood using FACS Calibur flow cytometry (Becton, Dickinson and Company, U.S.A).In the single administration study, hUC-MSCs were labeled with near-infrared dye DIR, and the metabolic characteristics of hESC-MSCs in the blood of cynomolgus monkeys were continuously and dynamically detected by flow cytometry.
In addition, peripheral blood of 300 μL was collected at before, 0.083 h, 0.5 h, 1 h, 3 h, 1, 3, 5, and 7 days post the first administration, as well as before, 0.083 h, 0.5 h, 1 h, 3 h, 1, 3, 5, 7, 10, 14, and 29 days post the final administration.Tissue samples of blood, heart, liver, spleen, lungs, kidneys, brain, testes, epididymides, uterus, ovaries, stomach, intestine, fat, and skeletal muscle were collected, and the distribution of hUC-MSCs in blood was detected by Q-PCR method.DNA is extracted from various tissues using a blood and tissue kit (Qiagen, U.S.) and the concentration of elution DNA is adjusted to the actual range.The upstream and downstream primer sequences are "TGT CTT TTT CTT GTT TTG GAG GAA " and "CCA GAC CAC CCA TAA TCT TGTGT", respectively, and the probe sequences are "AGG AGC CCA TCG GG".The primers above were designed and synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.PCR reaction conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.In this study, we used a TaqMan assay, in which a pair of primers and a specific fluorescent oligonucleotide probe was used.For each sample, three replicates were analyzed and the average of these replicates was calculated.Prior to PCR assay, DNA concentrations were measured with NanoDrop One to obtain genomic DNA content, and these concentrations were used to calculate the number of gene copies per nanogram of genomic DNA.
Animals were anesthesia by 35 mg/kg pentobarbital sodium, blood collection and necropsy were performed in a specific order (the Control Group, followed by the Low-dose Group and High-dose Group) to minimize potential confounders.

Statistical analysis
All data are presented as the mean ± standard deviation (SD) of n values, where n corresponds to the number of mice used.Statistical analyses were performed using one-way ANOVA, followed by Dunnett's test for comparisons to the Control group.Figures were generated using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA).Statistical significance was determined using SPSS (ver.26), with p < 0.05 considered significantly different.

Ethical statement
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Center for Safety

Bio-distribution of hUC-MSCs
Bio-distribution data is a crucial piece of evidence in elucidating the safety of hUC-MSCs for clinical use.To investigate the distribution characteristics after re-infusion, a method combining in vitro labeling with nearinfrared dye DIR and in vivo flow cytometry detection was established.It presents the advantages of simplicity, real-time, and rapid in the dynamic monitoring of stem cells in peripheral blood.Taking the percentage of hUC-MSCs in the blood as the detection index, the amount of hUC-MSCs reached a small peak at 5 min after administration, then gradually decreased, and began to rise at 1 h, reached the peak on 1 d after administration, and gradually decreased afterwards (Fig. 3).
In the toxicity study, the gene copy of hUC-MSCs could only be detected in the peripheral blood of animals in High-dose group at 30 min and 1 h post the initial administration, and reached the peak value at 0.5 h (5.35 copies/ng ± 2.21 copies/ng); after the final administration, the gene copeis of hUC-MSCs were only be detected in the peripheral blood of animals in High-dose group at 0.5 h, 1 h and 3 h post-administration, and reached the peak value at 0.5 h (4.78 copies/ng ± 2.17 copies/ng).See Fig. 3 for details.The gene copies of hUC-MSCs were all below the lower limit of quantification in all other tissues in all the animals.

General toxicity
During the study period, no abnormal symptom was observed in all the animals in the solvent control group and the low dose group.Facture on the left upper limb was observed in a high dose female animal (FM303) from 8 to 23 days post the initial administration.Redness at the injection site was observed in all the groups and the incidence was 80% in solvent control group, 80% in low dose group and 90% in high dose group, and all the animals were recovered afterwards.
Compared with the Control Group, no difference was found in averaged body masses of Low-dose group and High-dose group during the study period (Fig. 4).It is considered that the infusing of hUC-MSCs had no effect on the body mass of monkeys.During the study period, the body temperature of animals basically fluctuated within the normal range (around 37.5-39.5 °C for cynomolgus monkeys), and no difference was found comparing with the Control Group (Fig. 4).Blood pressure and electrocardiogram values of all the cynomolgus monkeys were also within the normal range, as no obvious difference associated with the administration of hUC-MSCs were observed (Fig. 5).

Hematology, serum biochemistry and urinalysis
Hematology and Serum Biochemistry data of animals administered hUC-MSCs were demonstrated in Figures 6  and 7, respectively.On D17, the levels of WBC, Lymph, Baso in the High-dose group were significantly higher than that in the Control group, Baso in the Low-dose group was significantly higher than that in the Control group.On R1, Lymph in both Low-dose group and High-dose group were significantly higher than that in the Control group, and the increase of Lymph was not observed by the end of recovery period.
CRE and GLU in the High-dose group were significantly higher than that in the Control group on D3.C3 in the High-dose group was significantly higher than that in the Control group on D17.LDH in both Low-dose group and High-dose group were significantly higher than that in the Control group on R1.GLU in the Highdose group were significantly higher than that in the Control group on R1.No change on other hematological, serum biochemistry indexes as well as the uinalysis indexes of animals administered hUC-MSCs was found.

Immunotoxicity and immunogenicity
The changes on the T lymphocytes count were summarized in Fig. 8. CD3 + /CD4 + T lymphocyte in both the Low-dose group and High-dose group were significantly higher than that in the Control group on R29.This change had no biological significance, as it is mainly attributed to the decrease in the Control group.On D3, D17, and D29, the count of Treg cell (CD25 + Foxp3 + ) in the High-dose group was significantly lower than that in the Control group.The cells used in this study were human-derived stem cells.The above changes suggested that the monkeys developed xenogeneic immune rejection.
IL-6 in the High-dose group was significantly higher than that in the Control group on D29 (Fig. 8).Combined with the overall data, this difference might due to the individual differences of animals.No other change on the cytokine profile was found.
Anti-hUC-MSCs antibodies were failed to be detected in any monkey on D3, D8 and D15.By end of the administration (D29), the antibodies were found in 9 animals in both Low-dose group and High-dose group.Further, by end of the recovery period (R29), the antibodies were detected all the animals in both Low-dose group and High-dose group.A certain time-effect relationship of the generation of antibodies was observed (Table 1).

Histopathology
By the end of the dosing perid, absolute and relative liver mass in the High-dose group was increased compared to Control group, and relative spleen mass in the High-dose group was increased compared to Control group (Fig. 9).These lesions were recovered by the end of recovery period.Although histopathological examination showed that no changes associated with the administration of hUC-MSCs were observed in the lungs and spleens, the changes of the absolute and relative lungs masses in the High-dose group may relate to the bio-distribution of hUC-MSCs.The changes of relative spleen mass may relate to the immune rejection of hUC-MSCs on xenogeneic animals.
In High-dose group, the thymus gland volume was found reduced in two animals.Histopathological examination revealed a mild to moderate decrease in lymphocytes in the thymic cortex (Fig. 10A,B).Meanwhile, a mild decrease in lymphocytes in the thymic cortex in one monkey in Control group (Fig. 10C).There was very mild increase of macrophage in the spleen white pulp in some animals of High-dose group.There was also very mild increase macrophage in the spleen white pulp in some animals in Control group.A mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermisin dermal or dermal-subcutaneous site in each group (Fig. 10D-F).

Discussion
Stem cells are undifferentiated cells possessing different degrees of self-renewal and multidirectional differentiation potential.Stem cells can proliferate in an undifferentiated state in vitro and differentiate into specific functional cells 22 .Currently, stem cells have shown promise in the treatment of malignant tumors, hematological diseases, neurological diseases, autoimmune diseases, metabolic diseases, cardiac diseases, ophthalmic diseases  and skin diseases [23][24][25][26] .However, the toxicity risks of stem cells have received wide attention, and sufficient preclinical safety research is a necessity for providing safety data and ensuring the safety of human use in clinical trials.Currently, there are few reports on the distribution and migration of hUC-MSCs in vivo.To understand the distribution characteristics of hUC-MSCs in vivo after cell transplantation, we used the fluorescent dye DiR to label hUC-MSCs before infusioninto monkeys.Flow cytometry method was used to detect the expression of DIR-labeled hUC-MSCs in the peripheral blood of monkey.DiR is a fluorescent dye with fluorescence located in the near-infrared region and could insert two 18-carbon chains into the cell membrane to form a stable label [27][28][29][30] .Despite that the detection period for dye labeling tracer is relatively short, it is a convenient, non-invasion method, and allows a real-time dynamic imaging.As demonstrated in our previous study (data not shown), both in vivo imaging and immunofluorescence method were simultaneously applied to detect the distribution and metabolism of UC-MSC in mice.The DiR could provide sensitive and reliable information for at least 2 weeks.Data obtained from the two methods were mutually verified, and together made the data more convincing.The DiR could stably label MSCs without an obvious effect on the biological characteristics of the cells, including morphology, viability, cell cycle and cell proliferation.We achieved dynamic detection of hUC-MSCs in the blood of monkey via flow cytometry.Our data showed that hUC-MSCs reached a small peak 0.083 h after dosing, and the number of cells in the blood gradually reached the peak after 1 day.These results indicted that after intravenous injection, some hUC-MSCs were distributed throughout the body within 0.5 h, while most of the cells accumulated in the lungs along with the systemic blood circulation, and were subsequently released into the blood.The number of cells in the blood slowly diminished after 2 days of dosing, due to the metabolic apoptosis.
Subsequent to 5 repeated administrations of hUC-MSCs, only a few animals in the High-dose group showed low copies of hUC-MSCs in peripheral blood at 0.5 h to 1 h and 0.5 h to 3 h after the first and the 5th administration, respectively.At the other time points, stem cell gene copy was not detected in peripheral blood and tissues.There was no significant increase in the gene copy number in the peripheral blood of patients by the end of treatment compared with the mesenchymal stem cell.The proposed clinical administration frequency of hUC-MSCs is once per week and may be repeatedly administered for four times.In this study, monkeys as non-human primates were used, and hUC-MSCs were administered intravenously once a week for 5 consecutive months to observe the toxicity and severity that might be caused by hUC-MSCs.Toxicity studies in animals is required to simulate the clinical dosing schedule to provide information for the design of clinical trials, including the optimal clinical dose, dosing period, route and frequency.Factors of great importance include the justifications of the animal, dose, and dosing schedule.Cynomolgus monkeys, as non-human primates, are genetically similar to humans, and abundant background data under physiological conditions have been accumulated.In terms of dose selection, the proposed clinical dose of hUC-MSCs is 1 × 10 6 cells/kg, which is equivalent to 2.6 × 10 6 cells/kg in monkey.Hence, 3 × 10 6 cells/kg and 2 × 10 7 cells/kg (approximately 8 times of the clinical dose) were used as the low dose and high dose respectively in this study.As previously reported, the MSCs were repeatedly administered to monkeys for 4 weeks (once per week) or 16 weeks (once per 2 weeks) at up to 2 × 10 7 cells/kg, and no severe toxicity was observed [31][32][33][34] .As demonstrated in our study, the monkeys were tolerated at both doses well.Intravenous injection has been proposed to be the best route for administrating hESC-MSCs to patients, which we also used here in the monkeys to avoid an alteration in efficacy caused by a different administration route.According to the number of times of clinical application, the frequency of drug administration was set as one time per week, and the drug was given at 0,1,2,3,4 weeks respectively.The recovery period is set at 4 weeks.
The administration of MSCs had no effect on clinical symptoms.Redness at the injection site was observed in some animals among groups, which could be recovered at recovery period.Histopathological examination confirmed that the above-mentioned redness at the injection site was related to mechanical stimulations of intravenous administration, and was not correlated with the direct effect of hUC-MSCs.hUC-MSCs reduced the number of Treg cells.This might related to the immunologic reactions of the allograft rejection.There are also no MSCs-related changes in body weight, body temperature, blood pressure, hematology, serum biochemistry, urinary analysis, and cytokines among groups.On D28 and R29, animals detected anti-hUC-MSCs antibodies in Low and High-dose groups.The timepoints for antibody production was in accordance with the general regularity that the body was induced to produce IgG antibodies upon antigen stimulation.Previous study has demonstrated that hUC-MSCs have certain immunogenicity in cynomolgus monkeys.By the end of the dosing period, absolute and relative liver masses were increased in the High-dose group compared to Control group, and relative spleen mass was increased in the High-dose group compared to Control group, which was recovered by the end of recovery period.Studies have reported that mesenchymal stem cell was distributed in varying proportions in the lungs and liver after transplantation [35][36][37] .Although no MSCs-associated histopathological lesions were found on lungs and spleen in High-dose group, the increased mass of the lungs and spleen might be related to the administration of hUC-MSCs.This effect on lungs might relate to the bio-distribution of hUC-MSCs.The increased mass of the spleen might relate to the xenogeneic immune rejection of hUC-MSCs.

Conclusion
In conclusion, hUC-MSCs at low and high doses had no significant effect on the clinical symptoms, injection site, body mass, body temperature, ECG, blood pressure, serum biochemistry, urinary biochemistry examination and cytokine levels of the animals.However, abnormal changes associates with the bio-distribution and xenogeneic immune rejection of hUC-MSCs were observed, including the decreased of Treg cells, mass change of lungs

Figure 2 .
Figure 2. The Flow cytometry test for anti-stem cell antibodies.Animal serum samples were collected at different time points, diluted and incubated with stem cells.Subsequent to the addition of labeled secondary antibodies, flow cytometry was used to detect the presence of anti-stem cell antibodies in the samples.

Figure 4 .
Figure 4. Changes on the body mass and temperature in monkey administered hUC-MSCs.Changes on the body mass (A) and body temperature (B) of monkeys during the study.(n = 10/group during dosing period; n = 4/group during recovery period).

Figure 6 .
Figure 6.Changes on leukocyte and related classification after repeated administration of hUC-MSCs.Averaged WBC (A), Neut (B), Lymph (C), Mono (D), Eos (E), and Baso (F) count during the study are shown.(n = 10/group during dosing period; n = 4/group during recovery period).Data are presented as means ± SD. */#P < 0.05, **P < 0.01.#represents significant difference compared to Control group and High-dose group; *represents significant difference compared to Control group and Low-dose group.

Figure 7 .
Figure 7. Changes on serum biochemical indexes after repeated administration of hUC-MSCs.Averaged levels of CRE (A), GLU (B), LDH (C), and C3 (D) during the study are shown.(n = 10/group during dosing period; n = 4/group during recovery period).Data are presented as means ± SD. */#P < 0.05, **P < 0.01.#represents significant difference compared to Control group and High-dose group; *represents significant difference compared to Control group and Low-dose group.

Figure 8 .
Figure 8. Changes on T lymphocytecount and IL-6 levels after repeated administration of hUC-MSCs.The percentage of CD3 + CD4 + T lymphocytes (A), CD3 + CD8 + T lymphocytes (B), CD4/CD8 (C) ratios and the percentage of CD4 + CD25 + Foxp3 + regulatory T lymphocytes (D) in peripheral blood, and levels of IL-6 in serum (E).Data are presented as means ± SD (n = 10/group in dosing period; n = 4/group in recovery period).*/#P < 0.05, **P < 0.01.#represents significant difference compared to Control group and High-dose group; *represents significant difference compared to Control group and Low-dose group.

Figure 9 .
Figure 9. Organ mass measured by the end of dosing period and recovery period.(A) Absolute organ mass by end of dosing period.(B) Relative organ mass by end of dosing period.(C) Absolute organ mass by end of recovery period.(D) Relative organ mass by end of recovery period.Data are presented as means ± SD (n = 6 monkeys/group in treatment period; n = 4/group for recovery period).*P < 0.05, **P < 0.01.*represents significant difference compared to Control group and Low-dose group.

Figure 10 .
Figure 10.Histopatholoic changes in thymus and injection sites.(A,B) A mild to moderate decrease in lymphocytes in the thymic cortex in high dose group (4×).(C) A a mild decrease in lymphocytes in the thymic cortex in vehicle control group (4×).(D) Normal injection site (10×).(E) A mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermis in dermal or dermal-subcutaneous site in vehicle control group (10×).(F) A mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermis in dermal or dermal-subcutaneous site in high dose group (10×).
Evaluation of Drugs (NCSED, IACUC approval No. IACUC-2018-K001), in compliance with the IACUC Constitution of NCSED, the Guide for the Care and Use of Laboratory Animals (https:// grants.nih.gov/ grants/ olaw/ Guide-for-the-Care-and-use-of-labor atory-anima ls.pdf) and AAALAC International's Position Statement.All study protocols (including the research question, key design features, and analysis plan) were prepared before the study and archived at National Center for Safety Evaluation of Drugs.

Table 1 .
Serum anti-hUC-MSCs binding antibodies detection results in monkey.