define mesenchymal stem cell from its fate:biodisposition of … · 2021. 8. 9. · 1 define...

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Define mesenchymal stem cell from its fate:

Biodisposition of human mesenchymal stem cells in normal and

Con-A induced liver injury mice

Li Han1,2

, Chenhui Ma1,2

, Huige Peng1, Zhitao Wu

1,5, Huiming, Xu

3, Jiajun Wu

1,2,

Ning Zhang1, Qinghui Jiang

1,2, Chen Ma

1,2, Ruimin Huang

1,2, Hai Li

4* and

Guoyu Pan1,2,*

1 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 201203,

Shanghai, China,

2 University of Chinese Academy of Sciences, 100049, Beijing, China

3 State Key Laboratory of Oncogenes and Related Genes, Renji-Med-X Clinical Stem

Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong

University, 200127, Shanghai, China.

4 Department of Gastroenterology, Renji Hospital, Shanghai Jiao Tong University

School of Medicine, 200127, Shanghai, China.

5 Nanjing University of Chinese Medicine, 210029, Nanjing, China

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 9, 2021 as DOI: 10.1124/jpet.121.000607

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Running title:Pharmacokinetics of mesenchymal stem cell

*Corresponding author:

Hai Li, Department of Gastroenterology, Renji Hospital, Shanghai Jiao Tong

University School of Medicine, Pujian Road 160, Shanghai 200127, (China). E-Mail:

[email protected]

Guoyu Pan, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,

Haike Road 501, Shanghai 201203, (China), Tel. +86-21-20231000-1715, E-Mail:

[email protected]

Text pages: 35

Tables: 3 (Supplemental: 1)

Figures: 5 (Supplemental: 7)

References: 40

Words in Abstract: 234

Words in Introduction: 506

Words in Discussion: 1338


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mailto:[email protected]

mailto:[email protected]


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ABBREVIATIONS: ALT (Alanine aminotransferase); ANOVA (analysis of

variance); AST (aspartate aminotransferase); AUC(area under the concentration-time

curve); Cmax (maximum concentration ); Concanavalin A (Con A); hMSC(human

mesenchymal stem cell); mMSC (mice mesenchymal stem cell); i.v. (intravenous);

IVIS (in vivo imaging system); qPCR (quantitative polymerase chain reaction); PBS

(Phosphate buffered saline); PK (pharmacokinetics); SD (standard deviation); t1/2,

half-life; Tmax, peak time.


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Abstract

The pharmaceutical industry and clinical trials have been revolutionized

mesenchymal stem cell-based therapeutics. However, little has the pharmacokinetics

of transplanted cells been characterized in their target tissues under healthy or disease

condition. A qPCR analytical method with matrix effect was developed to track the

biodistribution of human mesenchymal stem cells in normal and Con A induced liver

injury mice. MSC disposition in blood and different organs were compared and

relevant PK parameters were calculated. Human and mice MSCs (hMSCs and

mMSCs) displayed a very similar pharmacokinetic profile in all tested doses: about 95%

of the detected hMSCs accumulated in the lung, 3% in the liver, while almost

negligible cells were detected in other tissues. A significant double peak of hMSCs

concentration emerged in the lung within 1-2 hours after intravenous injection, so did

the mMSCs. Prazosin, a vasodilator could eliminate the second peak in the lung and

increase its Cmax and AUC by 10% in the first 2 hours. The injury caused by Con A

was significantly reduced by hMSCs while the Cmax and AUC0-8 of cells in the injured

liver was decreased by 54% and 50% respectively. And the Cmax and AUC would be

improved with the alleviation of congestion through the administration of heparin.

The study provides a novel insight into the pharmacokinetics of exogenous MSCs in

normal and Con-A induced liver injury mice which provided a framework for

optimizing cell transplantation.


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Significance Statement

MSCs are known for their potential as regenerative therapies in treating several

diseases, while an insufficient understanding of the pharmacokinetics of MSCs

restricts their future application. The current study was the first time to elucidate the

PK and the possible factors including dosage, species and derived sources, disease in

a systematic way. Furthermore, we investigated that Con A-induced liver injury

significantly prevented cells from entering the injury site which could be reversed by

the diminished congestion achieved by heparin.


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Introduction

Since CAR-T therapies were approved by FDA, cell therapies have attracted

increasing attention for their excellent therapeutic potency. As a multipotent

progenitor cell product, mesenchymal stem/stromal cells (MSCs) are well-known for

their potential in regenerative medicine and disease treatments such as hepatitis and

liver failure (Ringden et al., 2006; Zhang et al., 2014; Lee et al., 2015; Shi et al.,

2017), Crohn's disease (Lopez-Santalla et al., 2020), myocardial infarction (Golpanian

et al., 2015). After hundreds of clinical trials conducted in different countries, the

positive results of Remestemcel-L (Mesoblast Ltd) for Graft-versus-host disease

(GvHD) at phase III of clinical test (Galipeau and Sensebe, 2018), implied that MSCs

could be the next promising cell product to be approved.

Although intensive studies have focused on the therapeutic benefits of MSCs,

insufficient information about the pharmacokinetic process also blurs its future

application. It is an essential but difficult task to estimate the contribution of MSCs

exposure to its efficacy (Brooks et al., 2018). However, there have been a lot of

conflicts towards the tissue distribution and residence time in vivo. For example,

Ryang Hwa Lee found that the venous administrated MSCs disappeared within 5 min,

about 83% of cells accumulated in the lungs, and less than 0.05% of cells remained in

mice after 48 hours (Lee et al., 2009). While Niyibizi reported that GFP-labeled


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MSCs could survive in mice lung up to 150 days (Niyibizi et al., 2004). The route of

administration also complicated the residence time of MSCs, and it was reported that

MSCs could live more than 5 months when they were injected into muscles (Gao et

al., 2001; Braid et al., 2018). Besides, it is still questionable whether MSC studies

should be conducted on immunodeficiency animal models. Since the survival time of

MSCs maybe tripled in such animals (Zangi et al., 2009), it’s less convincible to

transformed pre-clinical results from these animals into real clinical performance in

the future.

Collectively, there are several fundamental questions about MSCs in vivo distribution

that have not been systematically answered: as an “immune privileged” cell product

(Zhang et al., 2015), is there a significant diversity in MSCs pharmacokinetic

behaviors among species? What is the relationship between dose and exposure in its

target organs? Could the pathological condition affect MSCs distribution and

clearance?

In this study, MSCs were labeled with hydrophilic dye and detected by an IVIS (in

vivo imaging system). Meanwhile, the conventional qPCR method was optimized by

considering matrix effects to estimate human MSCs disposition in mice organs

quantitatively. The pharmacokinetics of hMSCs and mMSCs were investigated

simultaneously to clarify the impact of species differences. A double peak of hMSCs

concentration curve in the lung was observed when the dosage exceeded 2x107

cells/kg, and the vasodilator Prazosin would eliminate the double peak and increase

hMSCs accumulation in the lung. Furthermore, we identified distinct PK properties in


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the Con A-induced acute hepatitis model with a significant decrease in Cmax and AUC

in the liver, and the potential reason might be liver congestion which could be

ameliorated by a co-administration of heparin.

Materials and methods

Materials

Concanavalin A and Pentobarbital sodium were purchased from Sigma (St Louis,

USA). The cell culture medium [α-minimal essential mediun(α-MEM)], Dulbecco’s

modified Eagle’s medium (DMEM) containing low glucose(1g/L), penicillin and

streptomycin (P/S, 100×), fetal bovine serum (FBS) and 0.25%

Trypsin/ethylenediaminetetraacetic acid (EDTA) were obtained from Thermo Fisher

Scientific (Madison, USA). Glutamine (100×, 200mM) and Cy5-NHS were purchased

from Meilunbio (Dalian, China). Glycine was obtained from Sino Pharm Chemical

Reagent Co., Ltd. (Shanghai, China). Prazosin was purchased from Meryer Chemical

Technology Co., Ltd (Shanghai, China), and heparin was from J&K Chemical Ltd

(Shanghai, China).

Animal Study

Adult male C57/BL6 mice (6–8 weeks) were purchased from the Shanghai

Laboratory Animal Co. (Shanghai, China). All animals were maintained under the

SPF condition with a constant temperature (23 ± 1.5°C) and humidity (70% ± 20%)

on a 12 h light/dark cycle. All animal experiments and protocols were reviewed and


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approved by the Institute of Animal Care and Use Committee (IACUC) of the

Shanghai Institute of Material Medica, Chinese Academy of Sciences (Permit number:

2019-02-PGY-29).

In vivo transplantation and imaging of MSCs.

Mice were given 200 μl hMSCs or mMSCs (4×106, 1×10

7, 2×10

7 cells/kg) through

the tail vein. At the indicated time points (15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h),

mice were anesthetized with pentobarbital(60mg/kg) and sacrificed, anticoagulated

blood and selected organs were collected. Organs were washed by PBS and

fluorescence intensities were measured by an IVIS (Caliper Life Science, USA), IVIS

filter set: 640 ex/680 em. For prazosin experiments, mice were intraperitoneally

injected with prazosin (5 mg/kg) 30 minutes before MSCs. For heparin assay, mice

were injected intravenously with 800 U/kg heparin one hour before giving MSCs.

Establishment of murine Con A-induced hepatitis model

Male C57/BL6 mice were randomly divided into groups (n = 3 for each group): the

normal control, PBS, Con A group (10mg/kg) and Con A + hMSC (2×107 cells/kg)

group.

To examine the therapeutic effect, all mice were anesthetized with pentobarbital

(60mg/kg) and sacrificed, and blood and livers were harvested 24 h after Con A

challenged. Blood samples were collected and centrifuged (3,000 rpm, 4°C) for 15

min to obtain serum, and stored at -80°C for further analysis. Liver specimens were

dissected and placed in 10 % formaldehyde for subsequent histological analysis while


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the rest was stored at-80°C.

Hematoxylin–Eosin Staining

Liver tissues were randomly selected for pathology analysis. Hematoxylin-Eosin

(H&E) staining were performed on 5-μm-thick liver sections of Paraffin-embedded

formaldehyde-fixed tissues to evaluated for pathological changes.

Human MSC culture

Human MSCs were kindly provided by Prof Hai Li and Huiming, Xu (Renji Hospital,

Affiliated to Shanghai Jiaotong University School of Medicine). Human MSCs

(female) were prepared and identified as described in previous study(Zhong et al.,

2020). The umbilical cord was obtained from informed mother. The collection and use

of the umbilical cord were approved by the Institutional Ethical Review Committee of

Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of

Medicine. Umbilical cord tissues were cut and digested then attached to culture plates

individually, followed by the addition of α-MEM containing 10% FBS and 1% P/S,

and then incubated at 37°C with 5% CO2. Approximately 12 days later, the cells were

cultured on new plates for further expansion and the following experiments.

Mice adipose derived MSCs isolation and culture

Mice adipose derived MSCs were harvested from male C57/BL6 mice (25 g)

according to a previously published protocol as previously reported (Estes et al.,

2010). Primary mMSCs were cultured with DMEM supplemented with 10% FBS plus

1% glutamine and 1% P/S in 5% CO2 at 37°C.


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When the cell density reached 80%–90% confluence, the cells were washed twice

with PBS and harvested with 0.25% trypsin/EDTA. The detached cells were

centrifuged at 1000 rpm for 3 min. The resultant pellet was obtained by centrifugating

(1000rpm, 3min) and resuspended in a fresh medium for further use.

Cell labeled by Cy5-NHS

After collection, cells were washed by PBS (pH 7.4). Then the pellet was incubated

with Cy5-NHS (10 μM, pH 8.0) for 30 min. The Glycine (100 mM) was added to

terminate the reaction and then washed with PBS (pH 7.4) for 3 times.

Blood Biochemical Analyses

The ALT and AST levels in serum were measured by biochemical assay kits

(Jiancheng Bioengineering, Nanjing, China), according to the manufacturer’s

instructions.

Cell Viability Assay

MSCs were seeded into 96-well plates (3×105 cells/ml) for 24 h prior to the

experiment. After that, 10 μl CCK-8 (Yeasen Biotech, Shanghai, China) was added to

each well and incubated for 2 h at 37°C in dark place, and then, the absorbance at 450

nm wavelength was measured by a microplate reader (BioTek, USA).

Enzyme Linked Immunosorbent Assays (ELISAs)

The concentration of serum cytokines, including interferon-γ (IFN-γ), tumor necrosis

factor-α (TNF-α) and interleukin-6 (IL-6) were detected by the corresponding ELISA


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kits (Multi Sciences Biotech, China). All the performance were conducted according

to the manufacturer’s protocol. The absorbance was tested using a microplate reader

(BioTek, USA) at 450 nm and 570nm.

Genomic DNA extraction

Genomic DNA extraction kit (Magen, China) was used to extract gDNA from tissue

based on the manufacturer’s protocol. Briefly, about 10mg tissues were weighted and

then added to tubes with 220 μL lysis buffer and 10 μL proteinase K (20 mg/ml). The

tissue suspension was digested in a water bath at 55°C overnight. 10 μL RNase was

then added, followed by further incubation at 70°C for 10 min. The gDNA was

purified and eluted following the manufacturer’s protocol.

Human-specific Alu qPCR primers

Quantitative PCR primers were designed targeting the human-specific unique

sequence of the Alu (Thi Tran and Kitami, 2019) and β-actin. The primer set for

human-specific Alu repeat was, forward, 5’-CTTGCAGTGAGCCGAGATT-3’;

reverse, 5’-GAGACGGAGTCTCGCTCTGTC-3’. The primer set for both human and

mouse actin was, forward, 5’-TCAGCAATGCCTGGGTACAT-3’; reverse,

5’-ATCACTATTGGCAACGAGCG-3’.

qPCR amplification

Quantitative real-time PCR (qPCR) assays were performed by Applied Biosystems

ABI 7500 System (Bio-Rad Laboratories, Hercules, USA) and the reaction mix was

loaded into 96-well PCR plates (Invitrogen, USA). The qPCR assay was performed in


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a volume of 20 μL that contained 10 μl qPCR SYBR Green Mix (Yeasen Biotech,

China), 1 μl forward and reverse primers and 200 ng template gDNA diluted in water.

The SYBR Green PCR reaction conditions were as follows: Hold stage was 95°C for

10 minutes, cycling was 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds.

Cell numbers-CT standard line establishment

Cells (106, 2.5×10

5, 6.25×10

4, 3.125×10

4, 7.8×10

3, 2×10

3, 500, 250, 125, 60) were

mixed with 10mg mice organ tissues to extract DNA. The standard curve of CT

values vs cell number (logarithmic form) can be obtained through QPCR

amplification. The number of hMSCs per unit weight in test tissues could be

calculated according to the standard curve.

For detecting the cells in blood, the fluorescent intensity of gradient-diluted Cy5

labeled MSCs (105, 5×10

4, 2.5×10

4, 1.25×10

4, 6.25×10

3, 3.13×10

3, 1.56×10

3, 780,

390 and 0) were mixed with 200μl anticoagulant blood measured at wavelength of

640 nm ex / 680 nm em, and the standard curve of fluorescent intensity- cell numbers

can be obtained. The calibration curve was constructed by linear regression of the

fluorescent intensity and cell numbers of standard samples. The number of hMSCs

per ml in test tissues could be calculated according to the standard curve

Statistical Analysis

GraphPad Prism 8.0 software was used for statistical analysis, and all data were

expressed as mean ± SD. Two tailed, unpaired t-test and one-way analysis of variance

(ANOVA) followed by Tukey’s multiple-comparison tests was used to analyze the


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two or more than two group comparisons, respectively. The effects of drug treatments

over time were analyzed using two-way ANOVA followed by Sidak’s

multiple-comparison tests. A P value < 0.05 was considered to be statistically

significant.

PK parameters including the maximum concentration (Cmax), the area under the

plasma concentration–time curve (AUC), elimination half-life, and mean residence

time were calculated by noncompartmental analysis with WinNonlin software

(version 6.2; Pharsight, Cary, NC).


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Results

Biodisposition of hMSCs in mice after i.v. administration

An imaging method for labeling cells with Cy5-NHS (Supp. Fig. 1 and Supp. Fig. 2)

and an optimized qPCR method (Supp. Fig. 3) were employed to detect hMSCs

distribution in mice respectively. Briefly, Cy5-NHS labeled hMSCs were injected

intravenously at various doses (4x106, 1x10

7, and 2x10

7 cells/kg). The animals were

anesthetized and then sacrificed at different time points and organs were collected.

The fluorescence intensities of the liver and lung were measured by IVIS.

The concentration curves of hMSCs in the lung and liver were displayed in Figure 1

respectively, and related PK parameters were listed in Table 1. The parameters were

calculated according to qPCR results. The half-life in the liver and lung was about 20

hours and 6 hours, respectively (Table 1). Near 95% of the detected hMSCs were

distributed in the lung and almost no cells were detectable in other organs (including

heart, spleen and kidney). And the fluorescent result were also detected (Supp. Table 1


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and Supp. Fig. 4), and the results were very close to qPCR.

The AUC of hMSCs in the liver increased with dose; however, its AUC in the lung

disproportionately rose at a higher dose, which was attributed by the double peak that

appeared in the lung at approximately 1 hour after administration (Figure 1). Human

MSC concentrations in blood were calculated and relevant parameters indicated that

the half-life was about 7 hours, and 80% of detected hMSCs in blood were cleared

within 8 hours (Supp. Fig. 4).

Comparison of hMSCs and mMSCs elimination process after intravenous

injection

More importantly, we asked whether the derived source made a difference in the

distribution of transplanted cells. To maximize the species differences, MSCs isolated

from mouse adipose tissue was used as an allogeneic control and investigated by the

IVIS method. The dose of 2×107 cells /kg was selected for subsequent comparison.

Interestingly, the elimination process of mMSCs and hMSCs were very similar in the

liver and lung (Figure 2).

Vasodilator Prazosin hydrochloride eliminated hMSCs “double peak” in the lung

Since the double peak in the PK curve was observed for the first time, we

hypothesized the possible reason might be the re-captured hMSCs in the pulmonary

capillaries. To investigate the impact of microvessel sizes on hMSCs disposition,

Prazosin, an alpha-adrenergic antagonist, was utilized to dilate blood vessels. Human

MSCs were intravenously administered 30 minutes later after intravenous injection of


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Prazosin (5 mg/kg). It was found that Prazosin could increase the accumulation of

hMSCs in the lungs (Figure 3A), more MSCs entered the lungs within 1 hour. hMSC

AUC and Cmax both increased by about 10% (Table 2). Although the cells

accumulated in the lung increased after giving Prazosin, there were not obvious

alterations in the lung clearance process and its distribution in the liver (Figure 3B).

Distribution of hMSCs under Con A-induced liver injury

Next, we examined the link between hMSC exposure in the liver and its efficacy in

the mice hepatitis model. At first, we examined its capacity to attenuate inflammation

and injury in Con A-induced hepatitis. hMSCs were administrated through the tail

vein four hours later after giving Concanavalin A (10 mg /kg). Next, the fluorescence

signals of various organs were measured (Figure 4A, 4B, Supp. Fig. 5) and the exact

cell amount was analyzed as well. Surprisingly, there was a significant reduction in

the liver during the acute inflammation stage and the AUC0-4 was approximately 56%

of the figure for healthy counterparts while more cells accumulated in the liver with a

134% increase in the concentration in 8h when the serum transaminase level began to

decrease (Table 3). Liver biomarkers including serum ALT and AST levels showed a

rapid significant decrease by hMSC as we predicted (Figure 4C, 4D). These results

suggested an impracticable role of the prevalent “homing” theory that MSCs is tended

to migrate to the injury site when comes to explain the phenomenon in Con A-induced

hepatitis.

Congestion caused by Con A-induced liver injury prevent MSCs from entering


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into the liver

Finally, the observed blood congestion in the liver was hypothesized as the possible

cause to discourage MSCs from entering the injury site. An anticoagulant drug,

heparin, was given at 800U/kg via tail vein injection into C57 mice 30 minutes before

hMSC injection. Next, the fluorescence signals of various organs were measured

(Figure 5A, 5B, Supp. Fig. 6). As we expected, the accumulation of hMSCs in the

liver was significantly augmented by heparin (Figure 5A): the Cmax in the liver

increased by about 30% at 4 hours, and Cmax returned to the same as the hMSC group

after 24 hours (Figure 5B). The increased hMSC amount in liver was connected with

the improvement of liver damages (Figure 5E). So did the decrease of liver ALT, AST

levels and the proinflammation factors (TNF-α,IFN-γ and IL-6) (Figure 5C, 5D and

Supp. Fig. 7). Taken together, these results indicated that congestion in the liver could

be the potential risk hinders the hepatic distribution of therapeutic MSCs in the Con

A-induced model.


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Discussion

There are numerous reports about the biological characteristics of stem cells and their

therapeutic effects, while few studies were conducted about their disposition and fate

in target organs. The purpose of this study is to characterize hMSCs distribution in

mice with optimized qPCR/IVIS methods and clarify the contribution of liver

hepatitis to hMSCs kinetics in the liver.

In order to record therapeutic cell residence time in vivo, multiple strategies have been

adapted including fluorescence (Braid et al., 2018), transgenic (Prigent et al., 2015;

Braid et al., 2018), isotopic (Ghazavi et al., 2017), magnetic irons (Liu and Ho, 2017),

and flow cytometry (Stuckey et al., 2006; Elman et al., 2014; Nguyen et al., 2014),


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while membrane dyes (PKH26, DiR) (Li et al., 2013; Vaegler et al., 2014) or nuclear

dyes (Hoechst 33342,DAPI) (Creane et al., 2017) are still the most conventional

labeling methods. However, these lipophilic dyes can bind noncovalently to the lipid

regions of target cell membranes (Jensen, 2012), and accumulate in the liver or spleen

due to their leakage from the damaged cells. On the contrary, the hydrophilic dye

Cy5- NHS could covalently bind to alive cells, and it didn’t show nonspecific binding

in the liver after the elimination of hMSCs in vivo (Supp. Fig. 1C).

To have a more precise understanding of the amounts of transplanted cells in organs,

an improved qPCR method with a verified human Alu gene sequence was employed.

In this study, in order to obtain hMSCs standard curves with matrix effect, different

amounts of cells were mixed with 10 mg mice tissue homogenates before DNA

extraction. The results indicated approximately 500 hMSC cells could be detected

from 10 mg mice tissue homogenates, which is equivalent to about 200 hMSC cells in

200000 mice cells (Supp. Fig. 3). Compared with previous studies (McBride et al.,

2003; Creane et al., 2017) which simply used mice DNA mixed with human DNA to

establish a standard curve (Yokoo et al., 2006; Shim et al., 2015), our optimized

method simulate the complexity of real detection environments since the matrix effect

was included.

Human MSC concentrations in different organs were measured at three doses.

Importantly, it was found that 90% of the hMSCs were stuck in the lungs, while 2%-3%

dwelled in livers within 15 min and a negligible amount stuck in other organs (<0.5%).

However, 95% of MSCs will be eliminated from the body within 24 h. Interestingly, a


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double-peak phenomenon was noticed in the time-concentration curve in the lung

after intravenous administration (Figure 3), which was never reported before

according to our knowledge. It was reported that MSCs could be captured by the

pulmonary capillaries because their size (20-30μm) is large than the capillaries (10-15

μm) (Fischer et al., 2009). Some hMSCs might be stuck in the capillaries during their

first circulation, and the rest free cells may be trapped later during the following

circulations, forming the second peak in the lung. To test this hypothesis, Prazosin

was utilized to dilate blood vessels and accelerate the migration of MSCs to deeper

capillary positions. The double peak disappeared, thus both the Cmax and AUC in the

lung increased significantly. Prazosin also promotes more stuck cells migrated to

other target tissue like the liver as we expected. Our results were opposed with Gao,

who claimed that vasodilator drugs could reduce cell entrapment in the lung (Gao et

al., 2001). However, they used 111

In-oxine labeled MSCs to trace radioactivity in the

lung 48 h after hMSCs administration. According to our experience, tracing

radio-labeled MSCs is not a good method to detect MSCs quantitatively because of

the short half-life and cytotoxicity of radioactive material.

One of the main concerns in current MSCs preclinical studies is the interspecies

variety. Here, mMSCs and hMSCs kinetics in mice were both investigated. In order to

maximize the variances, MSCs isolated from different tissues were utilized: mice

adipose-derived MSCs (mMSCs) were compared with human umbilical cord-derived

MSCs (hMSC). Interestingly, the in vivo PK pattern of two kinds of MSCs was quite

close (Figure 2), which implied that the species of cells wouldn’t influence the fate of


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MSCs in vivo greatly. As immunodeficiency animals is not obligatory and may not

reflect clinic situation correctly, so it is feasible to conduct hMSCs PK studies in

wildtype animals directly.

For conventional chemical compounds, it is well accepted that their PK profile may

be altered significantly due to disease. Here, Con A-induced immune liver injury was

employed to investigate the PK profile of MSCs under disease. The liver damage was

alleviated after hMSCs administration (Figure 4), which was consistent with previous

reports(Akla et al., 2012; Tamura et al., 2016). It was reported that more MSCs tends

to migrate to the injury sites to alleviate inflammation (Gao et al., 2016). However, in

this study, the AUC0-4 of hMSCs in the Con A-induced injury liver reduced 50%

compared with the control group. The phenomenon that fewer hMSCs could access

the liver under liver injury within the first 4 hours could not be explained by its

tendency in gathering in the injury location. In fact, the homing effect didn’t happen

until approximately 4-24h after hMSCs administration: the concentration of hMSCs in

the liver in the disease mice was 40% more than the normal group.

The discrepancy of hMSCs PK/PD suggested that disease might interfere with the

distribution of MSCs. Much congestion occurred in the liver after Con A

administration(Ye et al., 2018), which was also observed in our results (Figure 5), and

the congestion might prevent MSCs from entering the liver. When the congestion was

alleviated by heparin, the Cmax in the liver increased by about 30% resulting in further

lower liver inflammation (Figure 5). Our results also explained why a simple

increasing giving dosage may not always necessarily bring in enhanced therapeutical


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outcomes in some cases(Marino et al., 2008; Wu et al., 2008), because the distribution

of MSCs in the target site might be restricted by disease condition like congestion.

Pharmacokinetics research of MSCs under disease might help to found out adverse

factors on MSCs distribution and potential ways to improve the efficacy.

However, there are also some limitations in this study. Firstly, in our experiment, we

only performed the multi-dose of hMSC in male mice. While in presious studies, the

hMSC distribution showed no significant changes between male and female

mice(Creane et al., 2017). But releated multi-dose pharmacokinetics studies in female

are also needed to perform to analyze the impact of gender on the elimination pattern.

Secondly, because the study capacity of IVIS is limited and we have to measure each

tissue’s fluorescence in time, we couldn’t handle too many animals at each time point.

However, every study in our manuscript were conducted at least twice, some of them

were repeated in triplicate. So the result in this study is enough to support our

conclusion.

It is interesting to note that mMSC underwent similar eliminating process as hMSCs

in the mice. One possible reason is that our mMSCs had been cultured and expanded

in vitro. It was reported the usage of certain growth supplements and/or cytokines

would increase MSC immunological profiles (immunogenic MHC molecules, Class I

and II HLA antigens), which may accelerate the elimination of mMSCs in

mice(Huang et al., 2010; Salvadori et al., 2019; Wang et al., 2019) and made the PK

profile of hMSC and mMSC close to each other. Since the in vitro culture is inevitable

during MSC manufacture, it is reasonable to speculate that the difference of PK


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profile raised by allogeneic and xenogeneic MSC transplantation maybe not as

significant as expected before. And it is feasible to use hMSC in vitro PK profile to

predict its clinc cell fate. However, other possible explainations could not be

completely excluded and deserve further exploration. Another thing need to address is,

multiple dose of MSCs has been adopted in clinical trials(Zhang et al., 2012; Suk et

al., 2016; Kabat et al., 2020), which was not investigated in this paper. Therefore,

investigating the elimination pattern of MSC after multiple doses will benefit the field

in future as well.

Conclusion

In our present study, an optimized qPCR and IVIS-imaging methods were applied to

track hMSCs elimination in mice more accurately. It was found that the intravenously

injected cells mainly gathered in the lungs and more than 95% of cells were quickly

cleared within 24 h, which indicated that MSCs need to be modified to increase their

survival in vivo. The mechanisms of exogenous cells clearance were similar, so it is

feasible to refer in vivo PK results on mice to design clinic studies.

Finally, the congestion in the liver induced by Con A greatly prevent MSCs from


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entering the liver leading to the discrepancy of PK/PD, which showed the damage

induced by the disease may prevent cells from reaching the target site rather than

exerting its homing effects. The decreased cell distribution in the liver may be

reversed after the alleviation of congestion in the liver and benefit its therapeutic

effects remarkably. We believed that profound knowledge of the pharmacokinetics of

MSCs in disease is fundamental for the optimization of future transplantation and safe

application

Ethics statement

This study was conducted and followed the recommendations of the Institutional

Animal Care and Use Committee (IACUC), Shanghai Institute of Materia Medica

(SIMM). The protocol was approved by the Institutional Animal Care and Use

Committee (IACUC), Shanghai Institute of Materia Medica (SIMM).

The collection and use of the umbilical cord were approved by the Institutional

Ethical Review Committee of Shanghai Children’s Medical Center, Shanghai Jiao

Tong University School of Medicine.


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Author contribution

Participated in research design: Li Han, Chenhui Ma, Huige Peng, Zhitao Wu, Hai li,

Huimin Xu, Ruimin Huang and Guoyu Pan.

Conducted experiments: Li Han, Chenhui Ma , Jiajun Wu, Ning Zhang and Chen Ma.

Performed data analysis: Li Han and Chenhui Ma.

Contributed new reagents or analytic tools: Huiming Xu, Hai Li, Qinghui Jiang and

Ruimin Huang

Wrote or contributed to the writing of the manuscript: Li Han, Chenhui Ma and

Guoyu Pan

Conflicts of Interest

All the authors declare no conflict of interest. No author has an actual or perceived

conflict of interest with the contents of this article

Financial Disclosure

No author has an actual or perceived conflict of interest with the contents of this

article.

Funding

This study was supported by the “Organ Reconstruction and Manufacturing” Strategic

Priority Research Program of the Chinese Academy of Sciences (grant

XDA16020205), and the National Science Foundation of China (grant 81872927).


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Figure legends:

Figure 1 Pharmacokinetics of hMSCs in mice lung and liver by qPCR

The lung (A) and liver (B) were collected and human Alu repeat was analyzed by

qPCR assay after the mice were intravenously administrated hMSCs at dose of 4×106,

1×107, 2×10

7 cells/kg. The data were expressed as mean ± SD (n = 3), and the


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experiments were repeated for 2 times.

Figure 2 Pharmacokinetics of cy5-labelled mMSCs and hMSCs in C57 mice by IVIS

imaging assay

The fluorescence intensity of MSCs in mice lung (A) and liver (B) were measured by

IVIS after giving hMSCs or mMSCs to C57 mice at dose of 2×10

7 cells/kg. The data

were analyzed by Graphpad Prism software and expressed as mean ± SD (n = 3), and

the experiments were repeated for 2 times.

Figure 3 The cy5 labeled hMSCs pharmacokinetics in lung and liver after Prazosin

administration by IVIS imaging assay

The fluorescence of hMSCs in lung (A) and liver (B) were measured after the mice

were intravenously administrated Prazosin and hMSCs. *indicates P<0.05,

**indicates P<0.01 when compared to hMSC group and data were expressed as mean

± SD and statistical difference was determined by two-tailed t’test (n = 3), and the


Figure 4 The exposure and efficacy of hMSCs in mice with Con A-induced liver

injury

The hMSCs fluorescence intensities in the mice lung (A) and liver (B) were analyzed

by IVIS. The ALT and AST concentrations in mice blood measured to indicate liver

functions after hMSCs administration (C, D). *indicates P<0.05, ***indicates

P<0.005 when compared to hMSC group, and # indicates P<0.05, ## indicates

P<0.01, when compared to Con A group. The data were expressed as mean ± SD and


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statistical difference was determined by two-way ANOVA followed by Sidak’s

multiple-comparison tests (n = 3), and the experiments were repeated for 2 times.

Figure 5 Pre-treated heparin increased the exposure and efficacy of hMSCs in Con

A-induced liver injury mice

The hMSCs fluorescence intensities in the mice lung (A) and liver (B) were analyzed

by IVIS. The ALT and AST concentrations in mice serum measured to indicate liver

functions after hMSCs administration (C, D). Classical images of Control, Con A,

Con A + hMSC, and Con A + hMSC + Heparin groups were examined by H&E

staining, magnification ×20, Scale bar = 100μm. Symbols: black arrow--inflammation

infiltration, red rectangle—hemorrhage, blue circle—degeneration and necrosis. *

indicates P<0.05,** indicates P<0.01, *** indicates P<0.001 when compared to

control group and # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001

when compared to Con A group. $$$ indicates P<0.001 when compared to Con A +

Heparin group. %% indicates P<0.01 when compared to hMSC + Con A group. Data

were expressed as mean ± SD and statistical difference was determined by by

two-way ANOVA followed by Sidak’s multiple-comparison tests (n = 3), and the



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Table 1. Pharmacokinetic parameters in mice lung and liver after multi-dose hMSCs

administration

Unit

lung liver

Low Medium High Low Medium High

AUClast h×(Cells(108)/kg) 17.9±7.34 121±10.2 187±22.9 0.11 ±0.04 0.22 ±0.1 0.28±0.08


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Tmax h 0.25±0 0.25±0 0.75±0.43 0.25±0 0.58±0.38 0.33±0.14

Cmax Cells(107)/kg 48.2±4.28 135±35 237±86 0.28±0.01 0.39±0.03 0.95±0.05

MRT h 6.71±0.08 7.12±0.54 6.97±0.89 17.00±3.57 15.65±4.08 16.86±6.70

t1/2 h 4.68 ±1.05 5.71±1.21 5.77±1.16 16.79±7.64 18.61±9.77 22.17±7.84

AUClast, area under the concentration curve from zero to observed; Cmax, maximal observed concentration; MRT, mean residence

time; t1/2, elimination half-life, Tmax, peak time

Table 2. Pharmacokinetic parameters of hMSC in liver and lung after Prazosin

administration

unit

Liver Lung

hMSC

Prazosin

+hMSC

hMSC

Prazosin

+hMSC

AUClast0-2 h× FU(106) 193.4±14.4 228.4±16.3 746.4±38 999±40.3**


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AUClast2-8 h× FU(106) 419.9±34.7 660.3±67.6 2123±94.9 2612±151.4**

Tmax h 1 0.5 0.25 1

Cmax FU(106) 113.4±21.5 152.3±21.0 448.3±51.7 561.7±26.5*

AUC, area under the plasma concentration curve; FU, fluorescent units; Cmax, maximal observed concentration; MRT, mean

residence time; Tmax, peak time. *indicates P<0.05, **indicates P<0.01 when compared to hMSC group

Table 3. Pharmacokinetic parameters of hMSC in liver and lung between Con A

injury model and vehicle group

unit

Liver lung

hMSC

Con A

+ hMSC

hMSC

Con A

+ hMSC

AUClast0-4 h× FU(106) 174.6±15.83 90.23±12.02** 1841.00±153.30 1276.00±279.40*


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AUClast4-24 h× FU(106) 462.2±42 554.80±104.30 7825.00±575.50 7915.00±1651.00

Tmax h 2 2 2 2

Cmax FU(106) 59.53±8.26 33.33±8.32* 643.30±74.50 450.00±141.70

AUC, area under the plasma concentration curve; FU, fluorescent units; Cmax, maximal observed concentration; MRT, mean

residence time; Tmax.peak time. *indicates P<0.05, **indicates P<0.01 when compared to hMSC group


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