Mobilization of multilineage-differentiating stress-enduring cells into the peripheral blood in liver surgery.

PURPOSE: This study investigated whether liver damage severity relates to the mobilization of multilineage-differentiating stress-enduring (Muse) cells, which are endogenous reparative pluripotent stem cells, into the peripheral blood (PB) and whether the degree of mobilization relates to the recovery of liver volume following human liver surgery.
METHODS: Forty-seven patients who underwent liver surgery were included in the present study. PB-Muse cells were counted before surgery, on postoperative days (PODs) 3 and on POD 7. Liver volume was measured using computed tomography before and after surgery.
RESULTS: The PB-Muse cell count increased after surgery. The number of PB-Muse cells before surgery was higher, but without statistical significance in the group with neoplasms than in the healthy group that included liver donors (p = 0.065). Forty-seven patients who underwent liver surgery were divided into major hepatic resection (MHR; hepatectomy of three or more segments according to the Couinaud classification, n = 22) and minor hepatic resection (mhr; hepatectomy of two segments or less according to the Couinaud classification, n = 25) groups. PB-Muse cells increased at high rates among MHR patients (p = 0.033). Except for complication cases, PB-Muse cells increased at higher rates in the group with advanced liver volume recovery (p = 0.043). The predictive impact of the rate of increase in PB-Muse cells on the recovery of liver volume was demonstrated by multivariate analysis (OR 11.0, p = 0.014).
CONCLUSIONS: PB-Muse cell mobilization correlated with the volume of liver resection, suggesting that the PB-Muse cell number reflects the degree of liver injury. Given that the degree of PB-Muse cell mobilization was related to liver volume recovery, PB-Muse cells were suggested to contribute to liver regeneration, although this mechanism remains unclear.

Introduction

Liver surgery is associated with problems such as prolonged severe liver dysfunction after massive hepatectomy and postoperative liver failure. None of these conditions has infallible treatments. Preclinical studies using fatal cirrhosis and massive hepatectomy swine, rat, and mouse models have examined mesenchymal stem cell (MSC) transplantation to improve survival rates and liver function. Cell transplantation as a regenerative treatment is expected to eventually complement existing therapies for liver dysfunction [1].

Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative pluripotent stem cells that are stress tolerant [2]. They express genes relevant to pluripotency, such as Oct3/4, Nanog, and Sox2; can be identified as pluripotency surface marker stage specific embryonic antigen (SSEA)-3-positive cells in the bone marrow, peripheral blood (PB), and connective tissues of various organs; able to differentiate into triploblastic cells and self-renew at a single cell level while they are non-tumorigenic and exhibit low telomerase activity, consistent with the fat that they normally reside in the body [3-7]. Muse cells in the circulating blood can selectively home to damaged tissue by sensing sphingosine-1-phosphate (S1P), one of the general signals of tissue injury produced by phosphorylating the cell membrane component sphingosine in damaged cells, using S1P receptor 2 [8]. After homing to damaged tissue, Muse cells replace damaged/apoptotic cells by spontaneously differentiating into the same cell types as the damaged/apoptotic cells and repairing the tissue [8-11]. Therefore, they do not require gene introduction or differentiation induction for rendering pluripotency or a differentiated state. Furthermore, allogenic Muse cells can be used without human leucocyte antigen (HLA) matching or long-term immunosuppressant treatment, partly due to HLA-G expression implicated in immunotolerance of the placenta [8]. Clinical trials in which donor-derived Muse cells are delivered by intravenous infusion without HLA‐matching and immunosuppressant treatment to patients with acute myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, neonatal cerebral palsy, and amyotrophic lateral sclerosis are in progress, and their safety and efficacy have been reported in acute myocardial infarction and epidermolysis bullosa [12, 13].

Endogenous Muse cells may act as reparative stem cells through the above-described mechanisms. Suppose that extensive tissue damage is caused, the post‐infarct tissue produces S1P as a damage alert signal, thereby mobilizing endogenous Muse cells, probably from the bone marrow to the circulating blood; the Muse cells then home to the damaged tissue and repair the tissue. Clinical data support this hypothesis because the number of PB‐Muse cells increases after the onset of stroke and acute myocardial infarction [14, 15]. In acute myocardial infarction, patients with a high number of PB-Muse cells in the acute phase exhibit statistically meaningful cardiac function recovery, with less occurrence of heart failure at 6 months [15]. Thus, the number of endogenous Muse cells is a potential parameter of reparative activity.

Previous research on a physical hepatectomy mouse model showed that Muse cells accumulate only in the vicinity of damaged tissue, differentiating into multiple cell types that comprise the liver [16]. Similarly, data from an acute hepatitis mouse model confirmed that Muse cells migrate to damaged livers and differentiate into hepatocyte marker-positive cells [17]. This study investigated the dynamics of Muse cells to examine whether their mobilization is related to the severity of tissue damage and contributes to the recovery of liver volume after liver surgery in humans.

Materials and methods

Definitions

Based on the Couinaud classification, we defined major hepatic resection (MHR) as a hepatectomy of three or more segments, and minor hepatic resection (mhr) as a hepatectomy of two segments or less. Max PB-Muse was defined number of PB-Muse cells on postoperative day (POD) 3 or POD7, whichever was larger. Max AST was defined maximum aspartate aminotransferase on PODs 3 and 7. We defined the rate of increase in the PB-Muse cell count (Max PB-Muse /PB-Muse cell number before surgery) as ΔMuse, and the rate of increase in liver volume (liver volume on POD 7/ expected remnant liver volume after surgery) as ΔVolume. We graded postoperative morbidity according to the Clavien–Dindo classification [18], and postoperative mortality as any death occurring within 90 days of liver surgery.

Study protocol and patients

This prospective study recruited patients who were admitted to Iwate Medical University Hospital from October 2020 to August 2021 and underwent liver surgery. No exclusion criteria were used. The Ethics Committee of Iwate Medical University Hospital approved the study (reference MH2018-038). All patients provided their written informed consent before the study commenced, and the investigation conformed to the principles of the Declaration of Helsinki.

SSEA-3-positive Muse cell numbers in peripheral blood

The Muse cell number in the PB was measured based on SSEA3+ cell counts [7]. To quantify circulating SSEA3+ cells, 10 ml of PB was obtained per patient at each of three time points (before surgery and on PODs 3 and 7). Mononuclear cells were isolated by Lymphoprep™ (STEMCELL Technologies, Vancouver, BC, Canada) treatment. Anti-SSEA-3 antibody (1:100; BioLegend, San Diego, CA, USA) was added and incubated for 60 min in the dark at 4°C with gentle mixing. The cells were then washed three times with phosphate-buffered saline. Next, fluorescein isothiocyanate (FITC)-labeled anti-rat IgM antibody (1:200; Jackson Immunoresearch, West Grove, PA, USA) was added as a secondary antibody, and the cells were incubated and washed using the same method as the primary antibody. Finally, NucRed™ Live 647 (ThermoFisher, Waltham, MA, USA) was added for nuclear staining, and the cells were incubated for 15 min at room temperature. FACS (FACS Aria II™; Becton Dickinson, San Jose, CA, USA) and FACSDiva™ (Becton Dickinson) software were used for the analysis. Unincubated cells with no antibody, cells incubated with only secondary antibody (FITC-labeled anti-rat IgM antibody), and cells incubated with rat IgM (1:100; BioLegend) followed by the secondary antibody (FITC-labeled anti-rat IgM antibody) as an isotype control were used to determine nonspecific reactions and autofluorescence, as well as to set the gate for the SSEA-3+ cells [7].

Blood biochemical analysis

Blood samples underwent complete blood cell count and biochemical analyses for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil), and C-reactive protein (CRP) concentration and prothrombin time-international normalized ratio (PT-INR).

Liver volumetry

Computed tomography (CT) using a Synapse Vincent analyzer (Fujifilm Medical, Tokyo, Japan) was performed before and seven days after surgery to calculate liver volume. The expected remnant liver volume after surgery was estimated using preoperative CT measurements, and ΔVolume was calculated. A study on post-hepatectomy liver volume in 538 patients found that the greatest amount of liver regeneration occurred on POD 7. Also, the ΔVolume was ≥1.19 on POD 7 when more than 10% of the liver was resected [19]. Based on this, 1.19 was selected as the cut-off value for ΔVolume, and patients were divided into two groups: those with sufficient liver volume recovery (n = 22) and those with insufficient recovery (n = 25).

Statistical analysis

Data are presented as means ± standard deviations (SDs), and categorical variables are described as totals and frequencies. Data greater than +/- three times the SDs of the number of PB-Muse cells before surgery and Max PB-Muse were excluded as outliers. Differences in groups were assessed using the Mann-Whitney U test for continuous variables and a chi-squared or Fisher’s exact test (for expected counts of <5) for categorical variables. Correlations were analyzed using Spearman’s rank correlation. We performed multivariate logistic regression analysis using variables that had p-values <0.05 in the univariate analysis. The Youden index was used to determine the optimal cutoff value for the receiver operating characteristic curve (ROC). Statistical analysis was performed using JMP software version 14.2.0 (SAS Institute, Cary, North Carolina). Variables with p-values <0.05 were considered statistically significant.

Results

Characteristics and clinical features

This prospective study enrolled 59 patients who underwent liver surgery. Twelve cases were excluded because they were inappropriate for the study (one outlier case, five non-resection cases, and six data-deficient cases). Of the 47 patients finally included in the present study, 22 underwent MHR, and 25 underwent mhr. A significant difference was found between in the two groups in the number of HCC patients (Table 1). Surgical approach, operation time, blood loss, maximum white blood cells on PODs 3 and 7, maximum T-Bil on PODs 3 and 7, and length of hospital stay were significantly higher in the MHR group (Table 2). PB-Muse cells were also measured in 10 healthy subjects.

Table 1

Baseline characteristics.

	MHR (n = 22)	mhr (n = 25)	p-value
Age (years)	63.7 ± 14.6	66.4 ± 12.9	0.281
Sex			0.627
Male	18 (81.8%)	19 (76.0%)
Female	4 (18.2%)	6 (24.0%)
BMI (kg/m2)	23.5 ± 3.1	25.2 ± 4.9	0.138
Smoking	2 (9.1%)	7 (28.0%)	0.100
Alcohol intake	9 (40.9%)	8 (32.0%)	0.526
Chemotherapy	5 (22.7%)	9 (36.0%)	0.321
ASA			0.439
I	10 (45.5%)	7 (28.0%)
II	8 (36.4%)	13 (52.0%)
III	4 (18.2%)	5 (20.0%)
CCI	3.0 ± 2.7	2.2 ± 2.2	0.409
C-P class			0.926
A	21 (95.5%)	24 (96.0%)
B	1 (4.6%)	1 (4.0%)
HV			0.159
HBV	1 (4.6%)	4 (16.0%)
HCV	1 (4.6%)	4 (16.0%)
WBC (× 103/μL)	5.7 ± 1.6	6.1 ± 2.7	0.725
AST (U/L)	26.7 ± 15.2	28.9 ± 14.9	0.331
ALT (U/L)	25.7 ± 13.2	29.9 ± 22.6	0.907
T-Bil (mg/dL)	0.6 ± 0.3	0.6 ± 0.3	0.855
PT-INR	1.1 ± 0.1	1.1 ± 0.1	0.529
CRP (mg/dL)	0.7 ± 1.3	1.1 ± 2.2	0.438
Diagnosis
HCC	2 (9.1%)	11 (45.8%)	0.008
Metastasis	8 (36.4%)	8 (33.3%)	0.753
Other (malignant)	8 (36.4%)	5 (20.8%)	0.211
Benign	4(18.2%)	1 (4.0%)	0.116
Tumor diameter (mm)	47.6 ± 37.3	32.4 ± 26.9	0.093

Values are expressed as means ± SDs.

MHR, major hepatic resection; mhr, minor hepatic resection; BMI, body mass index; ASA, American Society of Anesthesiologists; CCI, Charlson comorbidity index; C-P, Child-Pugh score; HV, hepatitis virus; AST, aspartate aminotransferase; WBC, white blood cells; ALT, alanine aminotransferase; T-Bil, total bilirubin; PT-INR, prothrombin time-international normalized ratio; CRP, C-reactive protein; HCC, hepatocellular carcinoma.

Table 2

Comparison of surgical outcomes after major hepatic resection and minor hepatic resection.

	MHR (n = 22)	mhr (n = 25)	p-value
Surgical approach			0.038
LLR	15 (68.2%)	23 (92.0%)
Open	7 (31.8%)	2 (8.0%)
Multiple hepatectomy	4 (18.2%)	5 (20%)	0.874
Operation time (min)	280.1 ± 70.0	187.9 ± 66.9	0.001
Pringle maneuver (min)	54.6 ± 23.7	45.6 ± 43.1	0.397
Blood loss (ml)	279.2 ± 321.5	108.2 ± 153.7	0.002
Blood transfusion	1 (4.8%)	0 (0.0%)	0.270
Max. WBC (× 103/μL)	10.3 ± 2.5	9.2 ± 3.5	0.044
Max. AST (U/L)	472.5 ± 211.2	415.8 ± 276.1	0.228
Max. ALT (U/L)	412.8 ± 184.6	344.1 ± 228.3	0.186
Max. T-Bil (mg/dL)	2.0 ± 1.3	1.2 ± 0.7	0.005
Max. PT-INR	1.3 ± 0.1	1.2 ± 0.1	0.337
Max. CRP (mg/dL)	8.1 ± 5.7	10.2 ± 6.2	0.233
Morbidity Within seven days of surgery (Clavien-Dindo ≥ grade Ⅲ)	3 (13.6%)	2 (8.0%)	0.532
Bile leak	1 (4.5%)	1 (4.0%)
Pancreatic fistula	1 (4.5%)	0 (0.0%)
Pseudoaneurysm	1 (4.5%)	0 (0.0%)
Heart failure	0 (0.0%)	1 (4.0%)
Length of hospital stay (days)	19.4 ± 14.6	13.7 ± 10.5	0.004
Mortality	1 (4.6%)	0 (0.0%)	0.291

Values are expressed as means ± SDs.

MHR, major hepatic resection; mhr, minor hepatic resection; LLR, laparoscopic liver resection; max. WBC, maximum white blood cells on PODs 3 and 7; max. AST, maximum aspartate aminotransferase on PODs 3 and 7; max. ALT, maximum alanine aminotransferase on PODs 3 and 7; max. T-Bil, maximum total bilirubin on PODs 3 and 7; max. PT, maximum prothrombin time on PODs 3 and 7; max. CRP, maximum C-reactive protein on PODs 3 and 7.

Muse cell numbers in peripheral blood

Fig 1 shows the representative results of the quantification of Muse cells using FACS. The mononuclear cell fraction after Lymphoprep treatment was roughly selected by forward and side scatter gating (Fig 1A). The few remaining red blood cells, which were negative for NucRed Live 647, were removed by specific gravity centrifugation (Fig 1B). Nonspecifically labeled cells were assessed on the basis of the isotype control (Fig 1C). Finally, gating was set for the SSEA-3+ cells (Fig 1D). In each FACS analysis, the nonspecific bindings were assessed using isotype control, followed by the setting of the gate for the SSEA-3+ cells. The number of PB-Muse cells was expressed as cells per 100 μL of blood, as follows: the number of Muse cells (/100μL) = mononuclear cells (/100μL) × SSEA3+ cells (%).

Fig 1

How to count SSEA3 positive Muse cells using fluorescence-activated cell sorting (FACS).

A: The mononuclear cell fraction after Lymphoprep™ treatment was roughly selected by forward scatter and side scatter gating. B: The few remaining red blood cells (negative for NucRed™ Live647) were removed by specific gravity centrifugation. C: Nonspecifically labeled cells were removed based on the isotype control. D: The gating was set for SSEA-3+ cells.

We measured the number of PB-Muse cells before surgery in 59 cases and in an additional 10 healthy subjects (age, 29.6 ± 5.13 years; male-to-female ratio, 9:1). Among the 69 subjects, one case was excluded as an outlier (n = 68). The number of PB-Muse cells was higher, but without statistical significance, in the group with both benign and malignant neoplasms (426 ± 454.5 cells/100μL, n = 54) than in the healthy group, which included four liver donors (131.7 ± 160.2 cells/100μL, n = 14, p = 0.065; Fig 2A). In the group with both benign and malignant neoplasms (n = 54), the number of PB-Muse cells did not differ between the presence (415.1 ± 367.9 cells/100μL) and absence (434 ± 515.3 cells/100μL) of metastasis (p = 0.558, Fig 2B).

Fig 2

The number of PB-Muse cells before surgery.

A: Comparison of neoplasm group and healthy group. B: Comparison of presence and absent of metastasis.

The number of PB-Muse cells increased after surgery (before surgery 394.8 ± 458.8 cells/100μL, POD3 971.4 ± 1306.5 cells/100μL, POD7 645.9 ± 881.2 cells/100μL, Fig 3A). Within 7 days of surgery, five cases had grade 3 or higher complications according to the Clavien–Dindo classification (complication cases). In these cases, the number of PB-Muse cells did not differ before and after surgery (before surgery 416.4 ± 525.1 cells/100μL, POD3 193.6 ± 230.3 cells/100μL, POD7 220.8 ± 227.3 cells/100μL, Fig 3B). Except for the complication cases (no-complication cases), the number of PB-Muse cells increased and peaked on POD 3 (before surgery 392.2 ± 457.3 cells/100μL, POD3 1063.9 ± 1351.8 cells/100μL, POD7 696.5 ± 917.4 cells/100μL, Fig 3B). The rate of change from POD 3 to 7 (PB-Muse cell number on POD 7/PB-Muse cell number on POD 3) was higher, but without statistical significance in the complication cases (3.1 ± 5.5) than in the no-complication cases (1.1 ± 1.8, p = 0.168; Fig 3C).

Fig 3

Muse cell numbers in peripheral blood.

A: Changes in the Muse cell number in peripheral circulating blood after hepatectomy. B: Changes in the Muse cell number in peripheral circulating blood after hepatectomy for complication and no-complication cases. C: The rate of PB-Muse cell number changes from postoperative days (PODs) 3 to 7 (PB-Muse cell number on POD 7/PB-Muse cell number on POD 3) in complication and no-complication cases. *p <0.05.

Comparison of MHR and mhr

The number of PB-Muse cells before surgery did not significantly differ between the MHR (351.1 ± 456.5 cells/100μL) and mhr groups (433.2 ± 466.6 cells/100μL; Fig 4A). The Max PB-Muse was higher, but without statistical significance, in the MHR group (1,458.1 ± 1,567.9 cells/100μL) than in the mhr group (847.4 ± 1,047.9 cells/100μL, p = 0.079; Fig 4B). The ΔMuse was significantly higher in the MHR group (17.4 ± 33.9) than in the mhr group (4.6 ± 8.8, p = 0.033; Fig 4C).

Fig 4

Comparison of MHR and mhr.

A: The number of PB-Muse cells before surgery. B: Max PB-Muse in the MHR and mhr groups. C: ΔMuse in the MHR groups and mhr group. Max. Muse = maximum PB-Muse cell number on PODs 3 and 7.

Liver volumetry and Muse cell numbers

Of the 47 patients, 22 had ΔVolume ≥ 1.19, and 25 had ΔVolume < 1.19. ΔMuse did not differ between the two groups (ΔVolume ≥ 1.19 13.0 ± 22.9, ΔVolume < 1.19 8.4± 26.4, p = 0.241, Fig 5A), however, at the verification stage, except for the five cases with complications according to the Clavien–Dindo classification (grade 3 or higher within 7 days of surgery), ΔMuse was significantly higher in patients with ΔVolume ≥ 1.19 (16.3 ± 25.2) than in those with ΔVolume <1.19 (8.4 ± 26.4, p = 0.043; Fig 5B).

Fig 5

Liver volumetry and Muse cell numbers.

A: Comparison of ΔMuse between the Δvolume ≥1.19 and the Δvolume <1.19 groups. B: Comparison of ΔMuse between the Δvolume ≥1.19 and the Δvolume <1.19 groups for the no-complication cases.

Factors affecting ΔVolume

In the univariate analysis, many of the factors relating to ΔVolume were compared between the ΔVolume < 1.19 and ΔVolume ≥ 1.19 groups for the no-complication cases (Table 3). Among these factors, ΔMuse was significantly higher in the ΔVolume ≥ 1.19 group than in the ΔVolume < 1.19 group (p = 0.043). Significant differences in surgical approach were found between the two groups (p = <0.001; Table 3); therefore, we performed multivariate logistic regression analysis including these two factors. The ROC curve analysis of ΔMuse with respect to ΔVolume identified 6.0 as the optimal cut-off point [area under the ROC curve (AUC) 0.687, 95% confidence interval (CI) 0.514–0.859; Fig 6], with 52.9% sensitivity and 88.0% specificity. Even after adjusting for the surgical approach, ΔMuse was significantly higher in the ΔVolume ≥ 1.19 group than in the ΔVolume < 1.19 group (odds ratio 11.0, 95% CI 1.63–74.05, p = 0.014; Table 4).

Table 3

Comparison of the factors affecting Δ volume in no complication cases.

	Δvolume ≧ 1.19 (n = 17)	Δvolume < 1.19 (n = 25)	P-value
Age (years)	62.1 ± 13.4	65.9 ± 13.4	0.323
Sex			0.482
Male	12 (70.6%)	20 (80.0%)
Female	5 (29.4%)	5 (20.0%)
BMI, (kg/m2)	24.8 ± 3.4	24.4 ± 5.1	0.591
Smoking	2 (11.8%)	6 (24.0%)	0.322
Alcohol intake	4 (23.5%)	9 (36.0%)	0.391
DM	6 (35.3%)	7 (28.0%)	0.616
Liver cirrhosis	1 (5.9%)	0 (0.0%)	0.220
Chemotherapy	6 (35.3%)	8 (32.0%)	0.824
C-P class			0.232
A	17 (100.0%)	23 (92.0%)
B	0 (0.0%)	2 (8.0%)
Surgical approach			< 0.001
MHR	13 (76.5%)	6 (24.0%)
mhr	4 (23.5%)	19 (76.0%)
Max. Muse (cells/100μL)	1425.5 ± 1695.9	1109.0 ± 1134.0	0.530
ΔMuse	16.3 ± 25.2	8.4 ± 26.4	0.043
Pringle maneuver (min)	54.7 ± 25.1	47.0 ± 41.1	0.661

Values are expressed as means ± SD.

BMI, body mass index; DM, diabetes mellitus; C-P, Child–Pugh score; MHR, major hepatic resection; mhr, minor hepatic resection; max. Muse, maximum PB-Muse cell number on PODs 3 and 7.

Fig 6

Receiver operating characteristic curve analysis of the ΔMuse versus the Δvolume.

Table 4

Multivariate logistic regression analysis of factors associated with Δ volume.

Variables	Odds ratio	95% Confidence interval	P-value
Surgical approach (MHR or mhr)	13.0	2.30–73.54	0.004
ΔMuse (cut-off: 6.0)	11.0	1.63–74.05	0.014

Discussion

In this study, we evaluated the PB-Muse cell dynamics involved in human liver surgery. To evaluate the severity of tissue damage, we divided the 47 patients into the MHR and mhr groups. In the MHR group, as expected, operation time, blood loss, and length of hospital stay were significantly higher than in the mhr group (Table 2). Therefore, we regarded the MHR group as a more invasive group. Preoperative PB-Muse cell number varied considerably from case to case. We found that the number of preoperative PB-Muse cells was higher, but without statistical significance, in the group with both benign and malignant neoplasms (p = 0.065; Fig 2A). This outcome suggests that the presence of neoplasms affects preoperative PB-Muse cell number. Although smoking or drinking habits have been reported to influence Muse cell dynamics [14], our study showed no differences in preoperative PB-Muse cell number based on both traits (S1 and S2 Tables).

Our study revealed that PB-Muse cell mobilization was significantly higher after surgery than before-surgery (Fig 3A), Hence, PB-Muse cells were mobilized by surgical invasion. In the complication cases, the rate of change in the PB-Muse cell number from POD 3 to 7 was higher, but without statistical significance (Fig 3C). We also found that ΔMuse was significantly higher in the MHR group than in the mhr group (Fig 4C). These results demonstrate that PB-Muse cell mobilization depends on the resected liver volume and may also be triggered in response to complicating infections or circulatory disorders that cause tissue damage. Because Muse cell accumulation is specific to damaged tissue, this characteristic could be useful for objectively evaluating surgical invasion and quantifying damage severity.

S1P is a migration factor for Muse cells in patients with myocardial infarction [15]. We hypothesized that S1P would also act as a Muse cell migration factor following a hepatectomy. In the present study, the plasma S1P concentrations of 15 patients were analyzed; however, in our study, S1P did not exhibit the same dynamics as those reported for myocardial infarction in humans or other animals (S1 Appendix and S1 Fig). Previous studies have revealed the involvement of S1P in the proliferation, motility, morphology, and differentiation of tumor cells, neurons, vascular smooth muscle cells, and vascular endothelial cells; the protein is associated with five specific G protein-coupled receptors (S1PR1–5) [20-22]. In a study using a myocardial infarction rabbit model, S1PR2 expression was higher in Muse cells than in non-Muse cells. Inhibiting S1PR2 reduced the number of Muse cells engrafted in the left ventricle, increased the infarct range, and reduced the recovery of the left ventricular ejection fraction [8]. In vascular endothelial cells and bone marrow-derived cells, S1PR2 has an inhibitory effect on tumor angiogenesis and growth [23]. One confounding factor in our study was that 11 of the 15 patients (73.3%) who underwent S1P measurements had malignant disease; thus, our results may not exclusively reflect the effects of hepatectomy. A different experimental design and larger sample size will be needed to clarify the relationship between S1P and Muse cell mobilization in liver tissue damage.

ΔMuse was significantly different between the ΔVolume < 1.19 and ΔVolume ≥ 1.19 groups for the no-complication cases, even after adjustment for surgical approach (odds ratio 11.0, 95% CI 1.63–74.05, p = 0.014; Table 4). Liver regeneration after hepatectomy occurs primarily via hepatocyte hypertrophy [24]; however, the details of this mechanism and the potential involvement of non-liver cells remain poorly understood. In a study of a 70% hepatectomy swine model, administering porcine Muse cells via the portal vein improved jaundice and PT-INR [11]. Administering human bone marrow-derived MSCs and human bone marrow-derived Muse cells to a small-for-size rat model after liver transplantation showed that Muse cells migrated to the transplanted liver, and Ki-67-positive hepatocytes and sinusoidal endothelial cells were significantly higher in the Muse cell group. Furthermore, this study found that Muse cells could protect sinusoidal endothelial cells by expressing hepatocyte growth factor and vascular endothelial growth factor-A [25]. These studies suggest that Muse cells contribute to promoting liver function and regeneration. In the present study, in addition to the resected liver volume, known as a factor in liver volume recovery, the increase in PB-Muse cells may also be associated with post-hepatectomy liver volume recovery, suggesting a contribution to liver regeneration; however, this study could not verify the detailed mechanism of their involvement. A previous study using hepatectomy mouse models revealed that exogenously administered human Muse cells migrate and engraft to the injured liver and injured site, where they spontaneously differentiate into liver components, including hepatocytes; however, the frequency of human–mouse fusion cells is very low [16]. The role of Muse cells in liver volume recovery is likely to be functional, promoting tissue repair and liver regeneration rather than quantitative supplementation as trophic-like effects of MSCs [26].

The study had several limitations. First, the sample size may have been too small to statistically analyze many complex factors. Second, human clinical research lacks an established technique for labeling bone marrow-derived Muse cells, meaning that we could not perform detailed assessments of Muse cell engraftment, differentiation, and proliferation in liver tissue. Third, factors such as limitations on hospital stays meant that we could not improve the reliability of our conclusions by conducting a longer follow-up study. Fourth, we were not able to evaluate the relationship between PB-Muse cells and liver function because no fatal complications occurred during the measurement period, and only limited cases exhibited reduced liver function. Studies using animal models would provide further opportunities to establish a direct causal link.

In conclusion, PB-Muse cells are mobilized based on the resected liver volume, the degree of PB-Muse cell mobilization was related to liver volume recovery. Their concentration in PB can potentially be used to quantify the degree of surgical invasion. Furthermore, Muse cell mobilization could contribute to liver regeneration, although the detail of this mechanism remains poorly understood.

Plasma S1P concentration.

(DOCX)

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Plasma S1P concentrations and Muse cell numbers.

A: Changes in the number of PB-Muse cells after hepatectomy. B: Changes in plasma S1P concentrations after surgery. C: Plasma S1P levels (maximum S1P levels on PODs 1, 3, and 7) in the MHR and mhr groups. D: Relationship between plasma S1P levels and the Max PB-Muse. S1P = sphingosine-1-phosphate. *p <0.05.

(TIF)

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Comparison of the preoperative PB-Muse cell numbers affecting smoking.

(DOCX)

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Comparison of the preoperative PB-Muse cell numbers affecting drinking.

(DOCX)

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Effect size of analysis with significant difference.

(DOCX)

Click here for additional data file.

28 Mar 2022

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this article, Kikuchi et al. demonstrated the number of Muse cells in the peripheral blood is elevated after the liver surgery compared to the before surgery, particularly in patients with liver tumors and those who underwent major hepatic resection. The results reflect that the increase in Muse cells in the peripheral blood after the liver damage may facilitate liver repair, even though Muse cell differentiation and proliferation mechanisms are unclear.

Even the study is interesting, there are many concerns:

1. In blood loss, Max, T-Bil, Length of hospital stay (days) of table 2, figure 2A, 3A, 3B, 4B, 4C, 5A and 6B, the authors described that there are significant statistical differences. However, there seems to be no significant difference, since standard deviation is bigger than mean value. How the authors conducted statistical analysis? A column-scatter plot is recommended to reflect the distribution.

2. In diagnosis table 1 and surgical approach table 3, the authors described p-value. Please show which group there are significant difference.

3. Figure 1C: the gate p4 should be moved to the right to exclude a non-specific dot. The numbers of positive Muse cells should be calculated based on the newly set gate. Did the authors strictly set the gate according to the criteria of the FACS gate setting? The authors should check all the gate settings and re-analyse.

4. The authors define the number of peripheral blood Muse cells as follows; “The number of PB-Muse cells was expressed as cells per 100 ul of blood, as follows: number of PB-Muse cells (/100 ul)=mononuclear cells (/100 ul)×SSEA-3+ cells (%).” Have the authors investigated whether there is a change in the overall number of white blood cells due to liver resection or tumor formation?　 These basic data need to be added to the result section.

5. The authors discuss the study of S1P concentration in peripheral blood that “One confounding factor in our study was that 11 of the 15 patients (73.3%) who underwent S1P measurements had malignant disease; thus, our results may not exclusively reflect the effects of hepatectomy.”　Did the authors investigate whether there is a difference between S1P levels before hepatectomy and S1P levels in healthy subjects? If there is a difference, it would be one of the reasons for this discussion, so please add the data if you have it.

6. Basic information (age, gender, etc.) for healthy subjects is not listed. Please add this information to the results section.

7. Figure 2A: the authors mentioned “n=69” in figure 2A. According to the main text, the sample numbers of neoplasm are 55, and healthy numbers are 14. The “n=69” is too confusing and can be misinterpreted as 69 neoplasm samples plus 69 healthy samples used for analysis.

8. Although the authors showed in figure 3C that the ratio of POD7/POD3 is higher in complication cases, how to explain on POD3 in figure 3B that the Muse number is lower in complication cases than no complication cases?

9. p14 line1, what is the unit of “Δvolume”?

10. p15 line1, although table 2 is mentioned, "tumor diameter" is shown in table 1.

11. Regarding the same "tumor diameter", the p-value is shown as 0.06. Is this significant?

Reviewer #2: Muse cells are pluripotent non-tumorigenic cells with high capacity of tissue regeneration and function recovery. In addition, Muse cells are endogenous cells present in connective tissues (i.e. bone marrow, adipose tissue) that go into circulation (PB-Muse cells) to repair damage tissue when an injury occurs as it was demonstrated in patients undergoing cerebral stroke and acute myocardial infarction.

In this manuscript Kikuchi et al., analyzed Muse cells mobilization into the peripheral blood in patients with liver tumors that underwent major or minor hepatic resection (MHR and mhr, respectively) at days 3 and 7 post-surgery. The number of circulating Muse cells significantly increased in the MHR vs mhr patients. In addition, there was a positive correlation between circulating Muse cells and recovery of liver volume after surgery.

This is an interesting paper, with novel findings which could have implications in the process of liver regeneration after hepatectomy through the mobilization and repair capacity of endogenous Muse cells.

There are some issues that need to be clarified:

1- Fig. 3D and 3E show values of a patient with max Muse cell>10,000 and delta Muse >120 that look to be outliers. How was the liver recovery and AST values of this particular patient before and at days 3 and 7 after surgery?

2- Remove Fig 5 because of lack of power (only n=15). Include these results in Discussion as data not shown.

3- If S1P is not a major player in mobilization of Muse cells, what other mechanism(s) could be proposed?

Minor changes:

1- The terms maximum PB-Muse cell number and maximum AST are quite confusing, both need to be replaced.

2- Include in the reference section paper by Heneidi et al, 2013.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE revision.docx

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15 May 2022

Dr. Emily Chenette

Editor-in-Chief

Dr. Chengming Fan

Academic Editor

PLOS ONE

May 16, 2022

PONE-D-22-04405

Mobilization of multilineage-differentiating stress-enduring cells into the peripheral blood in liver surgery.

Thank you very much for the opportunity to revise our manuscript. The reviewer's comments were very helpful. We have corrected the manuscript point-by-point and have provided responses to each comment below. We believe that the manuscript has been substantially improved, and we would be grateful if you would consider its suitability for publication in PLOS ONE. We look forward to hearing from you.

Sincerely,

Koji Kikuchi M.D.

Department of Surgery,

Iwate Medical University School of Medicine,

2-1-1 Idai-dori, Yahaba, Iwate, 028-3609, Japan

Response to Editor

Comments to the Author

We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Reply:

Thank you very much for your suggestion. We have added this data to the Supporting Information files (S1 and S2 Tables).

Response to Reviewer #1:

Comments to the Author

In this article, Kikuchi et al. demonstrated the number of Muse cells in the peripheral blood is elevated after the liver surgery compared to the before surgery, particularly in patients with liver tumors and those who underwent major hepatic resection. The results reflect that the increase in Muse cells in the peripheral blood after the liver damage may facilitate liver repair, even though Muse cell differentiation and proliferation mechanisms are unclear.

Even the study is interesting, there are many concerns

1. In blood loss, Max, T-Bil, Length of hospital stay (days) of table 2, figure 2A, 3A, 3B, 4B, 4C, 5A and 6B, the authors described that there are significant statistical differences. However, there seems to be no significant difference, since standard deviation is bigger than mean value. How the authors conducted statistical analysis? A column-scatter plot is recommended to reflect the distribution.

Reply:

Thank you for your valuable comments. In blood loss, Max, T-Bil, Length of hospital stay (days) of table 2, figure 2A, 3A, 3B, 4B, 4C, 5A and 6B, differences in groups were assessed using the Mann-Whitney U test. The effect size of each analysis is shown in the Supporting Information file (S3 Table). The figures have been changed to a column scatter plot (Figs. 2A, 2B, 3A, 3B, 3C, 4A, 4B, 4C, 5A, 5B, S1A, S1B, and S1C).

2. In diagnosis table 1 and surgical approach table 3, the authors described p-value. Please show which group there are significant difference.

Reply:

Thank you for pointing this out. In Table 1, significantly more patients with HCC belonged to the mhr group. As you suggested, the description that was difficult to understand has been changed (Page 9, Lines 27). In Table 3, significantly more patients underwent MHR in the ΔVolume ≥ 1.19 group (Page 14, Lines 35).

3. Figure 1C: the gate p4 should be moved to the right to exclude a non-specific dot. The numbers of positive Muse cells should be calculated based on the newly set gate. Did the authors strictly set the gate according to the criteria of the FACS gate setting? The authors should check all the gate settings and re-analyse.

Reply:

Thank you very much for your invaluable comments. In each FACS analysis, the number of positive Muse cells was used to calculate the newly set gate on the basis of the isotype control. This information has been added in the revised manuscript (Page 10, Lines 36). We have checked all the gate settings, performed a reanalysis for some patients, and changed the figure (Fig. 1C).

4. The authors define the number of peripheral blood Muse cells as follows; “The number of PB-Muse cells was expressed as cells per 100 ul of blood, as follows: number of PB-Muse cells (/100 ul)=mononuclear cells (/100 ul)×SSEA-3+ cells (%).” Have the authors investigated whether there is a change in the overall number of white blood cells due to liver resection or tumor formation?　 These basic data need to be added to the result section.

Reply:

In accordance with your suggestion, we have added text and data on the change in the overall number of white blood cells in the Results section (Page 8, Lines 29), Table 1 (Page 9, Lines 20) and Table 2 (Page 10, Lines 13).

5. The authors discuss the study of S1P concentration in peripheral blood that “One confounding factor in our study was that 11 of the 15 patients (73.3%) who underwent S1P measurements had malignant disease; thus, our results may not exclusively reflect the effects of hepatectomy.”　Did the authors investigate whether there is a difference between S1P levels before hepatectomy and S1P levels in healthy subjects? If there is a difference, it would be one of the reasons for this discussion, so please add the data if you have it.

Reply:

Thank you for providing these insights. In the present study, we did not analyze S1P concentrations in healthy subjects. As you pointed out, if the S1P levels significantly differed between the pre-hepatectomy and healthy subjects, I think it is possible to consider something. This will be a subject for future research.

6. Basic information (age, gender, etc.) for healthy subjects is not listed. Please add this information to the results section.

Reply:

As suggested by the reviewer, we have added this data to the results section (Page 11, Lines 17).

7. Figure 2A: the authors mentioned “n=69” in figure 2A. According to the main text, the sample numbers of neoplasm are 55, and healthy numbers are 14. The “n=69” is too confusing and can be misinterpreted as 69 neoplasm samples plus 69 healthy samples used for analysis.

Reply:

We agree with your assessment. In Figure 2, the descriptions of n = 69 and n = 55 have been removed (Figs. 2A and B).

8. Although the authors showed in figure 3C that the ratio of POD7/POD3 is higher in complication cases, how to explain on POD3 in figure 3B that the Muse number is lower in complication cases than no complication cases?

Reply:

Thank you very much for your invaluable comments. As the number of complicated cases was as small as 6, it is difficult to give a definitive explanation. The complications were bile leak (onset at 2POD), bile leak (4POD), bile leak (7POD), pancreatic fistula (7POD), heart failure (3POD), and pseudoaneurysm (5POD). Assuming that Muse cell mobilization is suppressed at the onset of complications, this can be explained by the small number of Muse cells in complicated cases. In the process of repairing damaged tissue, the dynamics of Muse cells against further invasion such as complications is also an issue for future study.

9. p14 line1, what is the unit of “Δvolume”?

Reply:

Thank you for pointing this out. Since ΔVolume is a ratio, there is no unit like ΔMuse.

10. p15 line1, although table 2 is mentioned, "tumor diameter" is shown in table 1.

Reply:

Thank you for your comment. As you pointed out, this is a mistake. The content has been corrected (Page 8, Lines 27).

11. Regarding the same "tumor diameter", the p-value is shown as 0.06. Is this significant?

Reply:

Thank you for your comment. As you pointed out, this is a mistake. This is not significant. The content has been corrected (Page 8, Lines 27).

Response to Reviewer #2:

Comments to the Author

Muse cells are pluripotent non-tumorigenic cells with high capacity of tissue regeneration and function recovery. In addition, Muse cells are endogenous cells present in connective tissues (i.e. bone marrow, adipose tissue) that go into circulation (PB-Muse cells) to repair damage tissue when an injury occurs as it was demonstrated in patients undergoing cerebral stroke and acute myocardial infarction.

In this manuscript Kikuchi et al., analyzed Muse cells mobilization into the peripheral blood in patients with liver tumors that underwent major or minor hepatic resection (MHR and mhr, respectively) at days 3 and 7 post-surgery. The number of circulating Muse cells significantly increased in the MHR vs mhr patients. In addition, there was a positive correlation between circulating Muse cells and recovery of liver volume after surgery.

This is an interesting paper, with novel findings which could have implications in the process of liver regeneration after hepatectomy through the mobilization and repair capacity of endogenous Muse cells.

There are some issues that need to be clarified:

1- Fig. 3D and 3E show values of a patient with max Muse cell>10,000 and delta Muse >120 that look to be outliers. How was the liver recovery and AST values of this particular patient before and at days 3 and 7 after surgery?

Reply:

We thank the referee for the valuable comment. The patient underwent percutaneous transhepatic abscess drainage for liver abscess three times in the posterior segment before surgery. A follow-up CT scan showed that the abscess in the posterior segment had been replaced with a mass, and the patient was diagnosed with liver metastasis and underwent posterior segmentectomy. The ΔVolume in this patient was 0.93. The AST values were 26 (before surgery), 1200 (POD3), and 62 U/L (POD7).

2- Remove Fig 5 because of lack of power (only n=15). Include these results in Discussion as data not shown.

Reply:

Thank you very much for your important comments. We agree with you, and Fig. 5 has been removed because of the lack of power. However, this is the first report of the dynamics of S1P in liver surgery, and we consider it to be valuable data, despite its lack of power. Thus, we will show the data in the Supporting Information files (S1 Appendix and S1 Fig).

3- If S1P is not a major player in mobilization of Muse cells, what other mechanism(s) could be proposed?

Reply:

We appreciate the reviewer's comment on this point. No mechanism can be proposed at this time. The SDF-1-CXCR4 axis may also be a candidate factor that triggers Muse cell mobilization. As a result of animal experiments, however, it is said that the S1P-S1PR2 axis is more specific to Muse cell mobilization. Owing to the design of this study, it may be difficult for the number of Muse cells and S1P concentrations to increase. Although many animal experiments use fatal disease and complication models, in liver surgery, we determine the transection line to avoid congestion or ischemia in the remnant liver. That is, we perform surgery to minimize tissue damage. For this reason, it is possible that the mobilization of Muse cells and the S1P concentration were not noticeable in this study.

Minor changes:

1- The terms maximum PB-Muse cell number and maximum AST are quite confusing, both need to be replaced.

Reply:

In accordance with your suggestion, we have replaced these terms with shorter terms. Max PB-Muse was defined as the number of PB-Muse cells on postoperative day (POD) 3 or POD7, whichever was larger. Max AST was defined as the maximum aspartate aminotransferase on PODs 3 and 7. This information has been added in the revised manuscript (Page 5, Lines 25.)

2- Include in the reference section paper by Heneidi et al, 2013.

Reply:

As suggested by the reviewer, we have added this paper to the reference section (Page 20, Lines 20).

Submitted filename: Reply letter.docx

Click here for additional data file.

7 Jun 2022

29 Jun 2022

Dr. Emily Chenette

Editor-in-Chief

Dr. Chengming Fan

Academic Editor

PLOS ONE

June 29, 2022

PONE-D-22-04405

Mobilization of multilineage-differentiating stress-enduring cells into the peripheral blood in liver surgery.

Thank you very much for the opportunity to revise our manuscript. The reviewer's comments were very helpful. We have corrected the manuscript point-by-point and have provided responses to each comment below. We believe that the manuscript has been substantially improved, and we would be grateful if you would consider its suitability for publication in PLOS ONE. We look forward to hearing from you.

Sincerely,

Koji Kikuchi M.D.

Department of Surgery,

Iwate Medical University School of Medicine,

2-1-1 Idai-dori, Yahaba, Iwate, 028-3609, Japan

Response to Reviewer #1:

1. Since bar graphs were changed to plots, now the data became interpretable. On the other hand, values for average and SD overlapped with plots in the revised figures. For this, the referee asks authors to describe numerical values of average and SD in the main text, so that readers may understand the meaning of data. For example, page 11, second and third paragraphs, sentences for Fig 2 and Fig 3 needs numerical values for each group, neoplasm and healthy groups.

Reply:

In accordance with your suggestion, we have added numerical values for the average and SD in the Results section (Page 11, Lines 12), (Page 11, Lines 13), (Page 11, Lines 15), (Page 11, Lines 17), (Page 12, Lines 3), (Page 12, Lines 5), (Page 12, Lines 8), (Page 13, Lines 5), (Page 13, Lines 6), (Page 13, Lines 7), (Page 13, Lines 8), (Page 13, Lines 14) and (Page 13, Lines 17).

2. looking at Fig 2A and 2B, one single plot in neoplasm group seem to elevate the average in neoplasm group. They stated in the text that “the number of PB-Muse cell was higher in the group with both benign and malignant neoplasms (n =55) than in the healthy group, which included four liver donors (n = 14, p = 0.056; Fig. 2A).” Since there was no statistical difference, authors should not carelessly state higher. “High but without statistical significance” might be better sentence.

The same issue falls into other “higher” parts throughout the text.

Furthermore, authors should check the appropriateness of the single dot in the neoplastic/metastatic patients in Fig 2 to be included as a proper sample. This single dot seems to distorts the data that it should be.

Reply:

Thank you very much for your invaluable comments. We have changed the indicated sentences (Page 2, Lines 9), (Page 11, Lines 11), (Page 12, Lines 7), (Page 13, Lines 6), (Page 15, Lines 13) and (Page 16, Lines 7). In the case of the single dot in Fig. 2, that point has been excluded as an outlier because the number of PB-Muse cells before surgery was greater than +/- three times the SDs. We have added explanatory text regarding outliers in the Materials and methods section (Page 8, Lines 1) and Results section (Page 11, Lines 10).

3. page 11, third paragraph: why authors can conclude that POD3 was peak in Fig 3A. There was no statistical significance between POD3 and POD7 (p=ns). The same problem falls onto Fig 3B, as well. How POD7 can be the highest while POD3 and POD7 were ns.

Again a single plot in Fig 3C seems to be extraordinal case and thus is questionable to be included as one of the rational samples.

Reply:

As suggested by the reviewer, we cannot conclude that POD3 was a peak in Fig 3A and POD7 was the highest in Fig 3B. We have changed the indicated sentence (Page 11, Lines 17) and (Page 12, Lines 2). In the case of the single dot in Fig. 3C, that point has been excluded as an outlier because Max PB-Muse was greater than +/- three times the SDs. We have added explanatory text regarding outliers in the Materials and methods section (Page 8, Lines 1) and Results section (Page 8, Lines 13). We have also changed the data throughout the text after reanalysis.

4. page 13, top line: the MHR group (1,852.0 ± 2,431.8 cells/100μL) was higher than in the mhr group (847.4 ± 1,047.9 cells/100μL, p = 0.051; Fig. 4B -> higher but without statistical significance.

Reply:

In accordance with your suggestion, we have changed the indicated sentence (Page 13, Lines 6).

5. page 11: “ΔMuse was significantly higher in patients with ΔVolume ≥ 1.19 (16.3 ± 25.2) than in those with ΔVolume ˂1.19 (8.4 ± 26.4, p = 0.043; Fig. 5B). ΔMuse did not correlate with ΔVolume (p = 0.917; Fig. 5C).” -> The two sentences are inconsistent. Needs to be explained or reconsider the statement.

Reply:

Thank you for providing these insights. Although Fig. 5C showed a correlation, and not a relation between cause and effect, as you pointed out, the two sentences are too confusing. We have therefore removed Fig. 5C and the relevant sentence from the Results section and Discussion sections.

6. Table 2 is sloppy. Extra lines should be deleted.

Reply:

Thank you for your comment. As you pointed out, this is a mistake. The extra lines have been deleted.

Response to Reviewer #2:

1. Figures 3D/E have to be redone without including the outlier. P values needs to be re-calculated and results and discussion needs to modified accordingly.

Reply:

Thank you very much for your important comments. A case of Max PB-Muse greater than +/- three times SDs has been excluded as an outlier. We have performed a reanalysis that excluded this case. After this reanalysis, the Max PB-Muse and the ΔMuse did not correlate with the Max AST. Therefore, we have removed Fig. 3D and E and the relevant sentences from the Results section and Discussion sections.

Submitted filename: Reply letter.docx

Click here for additional data file.

6 Jul 2022

Mobilization of multilineage-differentiating stress-enduring cells into the peripheral blood in liver surgery: A cohort study

PONE-D-22-04405R2

Dear Dr. koji kikuchi ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Chengming Fan, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I think it could be accepted.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper has revised substantially. In the figures, the display of p-values is inconsistent. In some figures p value is not mentioned but simply indicated ns, and in other figures, the p-value is displayed numerically. Please unify to either.

Reviewer #2: Authors answered all my questions (results, figures, tables, discussion). Therefore MS is accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

12 Jul 2022

PONE-D-22-04405R2

Mobilization of Multilineage-Differentiating Stress-Enduring Cells into the peripheral blood in Liver Surgery

Dear Dr. Kikuchi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Chengming Fan

Academic Editor

PLOS ONE