1

Title:

2

Intravenous Infusion of Cardiac Progenitor Cells in Animal Models of Single Ventricular3

Physiology

45Authors:

6

Takuya Goto1, Daiki Ousaka1. Kenta Hirai 2, *Yasuhiro Kotani 1, Shingo Kasahara 1

7

Institutions:8

Department of Cardiovascular Surgery1 and Pediatrics2, Okayama University Graduate

9School of Medicine, Dentistry, and Pharmaceutical Sciences and Okayama University

10

Hospital, Okayama, Japan

11
12

Corresponding Author:

13

Yasuhiro Kotani, M.D., Ph.D.

14

Department of Cardiovascular Surgery,15

Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical

16

Sciences and Okayama University Hospital,

17

2-5-1 Shikata-cho, Kita-ku,

18

Okayama 700-8558, Japan

19

Tel.: +81-086-235-735

20

Fax: +81-086-235-7431

21

E-mail: yasuhiro.kotani@okayama-u.ac.jp

2223

Meeting Presentation: none

24
25

Word Count: 4987 words

1

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Visual abstract:27

Key question

28

What palliative stage is suitable for intravenous stem cell administration therapy in29single ventricular physiology?

30
31

Key findings

32

Intravenously injected stem cells were delivered to cardiac muscle in stage 1 and 2

33

palliation models in piglet.

34
35

Take-home message

36

Intravenous stem cell injection may be applicable in patients with single ventricular37

physiology before receiving Fontan type operation.

38
39
40
41

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42

ABSTRACT and KEY WORDS:

43

Objectives

44

This study aimed to identify the practical applications of intravenous cell therapy for45

single-ventricle physiology (SVP) by establishing experimental SVP models.

46

Methods

47

An SVP with a three-stage palliation was constructed in an acute swine model without

48

cardiopulmonary bypass. A modified Blalock–Taussig shunt (MBT) was created using

49

an aortopulmonary shunt with the superior and inferior vena cava (SVC and IVC,

50

respectively) connected to the left atrium (LA) (n=10). A bidirectional cavopulmonary

51

shunt (BCPS) was constructed using a graft between the IVC and LA with an SVC

52

cavopulmonary connection (n=10). The SVC and the IVC were connected to the53

pulmonary artery to establish a total cavopulmonary connection (TCPC, n=10). Half of54

the animal models were observed to examine their lifetimes. The other half and the55

biventricular sham control (n=5) were intravenously injected with cardiosphere-derived

56

cells (CDCs), and the cardiac retention of CDCs was assessed after 2 h.

57

Results

58

All SVP models died within 20 h. Perioperative mortality was higher in the BCPS group59

because of lower oxygen saturation (P<0.001). Cardiac retention of intravenously

60

delivered CDCs, as detected by magnetic resonance imaging and histology, was61

significantly higher in the MBT and BCPS groups than in the TCPC group (P<0.01).

62

Conclusions

63

Without the total right heart exclusion, stage-specific SVP models can be functionally64

constructed in pigs with stable outcomes. Intravenous CDC injections may be

65applicable in patients with SVP before TCPC completion, given that the initial lung66

trafficking is efficiently bypassed and sufficient systemic blood flow is supplied from the67

single ventricle.

3

68
69

Keywords

70

animal model, single ventricular physiology, cell therapy, cardiosphere-derived cell,71

intravenous

72

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73

Abbreviations and acronyms

74

BCPS: bidirectional cavopulmonary shunt; BV: biventricular; CDC(s): cardiosphere-75

derived cell(s); ePTFE: expanded polytetrafluoroethylene; IVC: inferior vena cava; LA:

76

left atrium; LV: left ventricle; MBT: modified Blalock–Taussig shunt; MRI: magnetic

77

resonance imaging; PA: pulmonary artery; SaO2: arterial oxygen saturation; SpaO2:

78

oxygen saturation in the pulmonary artery; SPIO: superparamagnetic iron oxide; SVC:

79

superior vena cava; SVP: single-ventricle physiology; TCPC: total cavopulmonary

80

connection

81
82

INTRODUCTION

83

Stem cell therapies have recently been shown to be feasible and safe interventions for84

treating patients with congenital heart diseases, such as dilated cardiomyopathy and85

single-ventricle lesions.1,2 Nevertheless, a variety of beneficial effects corresponding to86

the results of clinical trials have not yet been fully elucidated in appropriate

87

experimental models that precisely reflect in-vivo human pathophysiological conditions.

88

Experimental studies that recapitulate single-ventricle physiology (SVP) are extremely

89

challenging and technically difficult. No report has successfully achieved single-

90

ventricle circulation in small animals, and a large chronic animal model has not been91

established even for the use of ventricular assist devices in the postnatal heart. 3

92

Therefore, we aimed to establish novel animal models of single-ventricle circulation in93

pigs to assess surgical palliation-specific hemodynamics during the creation of single-

94

ventricle circulation, allowing the development of a new cell transplantation strategy

95

and outcome evaluation of stem cell distribution with respect to the route of delivery.

96
97

MATERIAL AND METHODS

98

Ethics Statement

5

99

The experimental protocol was approved by the Experimental Animal Committee of100

the Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical101

Sciences (approval number: OKU-2018941; approval date: March 27th, 2019). All

102

experimental animals were cared for in accordance with the institutional guidelines and103

the Guide for the Care and Use of Laboratory Animals.4

104
105

Study design and endpoint

106

Three-stage palliation models of the SVP were established to mimic circulation with

107

univentricular disease. These models included a modified Blalock–Taussig shunt, a108bidirectional cavopulmonary shunt, and a total cavopulmonary shunt model.

109

First, the hemodynamic features of five piglets of each model and control group were110

compared (n=5×4), and the lifetime of the SVP models after extubation was observed.

111

Next, piglets with biventricular (BV) circulation were compared using different stem cell

112

administration methods, and each single ventricular model with intravenous stem cell113

injection. This study included 35 pigs: a control group of five pigs, and two sets of three114

treatment groups, each including five pigs that shared a common control. Ten piglets

115

with BV circulation received intra-coronary injections of a medium (n=5) and stem cells

116(n=5). Five piglets received an intra-left ventricular injection of stem cells (n=5). Five117

piglets of each SVP model and the common control group (BV circulation) received

118

intravenous injections of stem cells (n=5×4). These seven groups were observed for 2

119

h under general anesthesia with artificial ventilation and sacrificed to extract their120

hearts. The hearts were examined using ex-vivo cardiac magnetic resonance imaging

121(MRI). Finally, the hearts of the three SVP model groups and the control group with122

intravenous injection were histologically analyzed.

123

The endpoint of lifetime observation was death.

6

124

The primary endpoint of this study was stem cell retention in the cardiac muscle after

125

intravenous injection.

126
127

Preparation of animal models

128

Eight-week-old Yorkshire pigs weighing 25 kg were anesthetized via inhalation of 2%

129

isoflurane. After median sternotomy and administration of 4000 units of heparin,

130

appropriate surgical procedure was performed. Normal Yorkshire pigs with BV hearts

131

underwent median sternotomy as a control for comparison.

132

To assess postoperative survival, the sternum was closed and extubated when133

adequate spontaneous respiration occurred.

134
135

Modified Blalock–Taussig shunt in a swine SVP model as a stage 1 palliation

136

model

137

After median sternotomy, the proximal part of the brachiocephalic artery was clamped

138

and a 6-mm diameter expanded polytetrafluoroethylene (ePTFE) tube was139

anastomosed in a side-to-end fashion using a continuous 6-0 polypropylene suture

140(Prolene, Ethicon, Raritan, NJ, USA). The distal end of the conduit was anastomosed141

to the main pulmonary artery (PA) in an end-to-side fashion under partial clamping of142

the PA, creating a direct systemic-to-pulmonary shunt (modified Blalock–Taussig

143

shunt, MBT; Figure 1A).

144

Venous blood flowing from the superior and inferior vena cava (SVC and IVC,

145

respectively) was drained into the left atrium (LA) by direct grafting of prosthetic

146

conduits in a Y-configuration with a 14-mm diameter ePTFE. Single-ventricular147

circulation was established by ligating the SVC and IVC at the atrial junction (Figure

148

1A).

149

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150

Bidirectional cavopulmonary shunt in a swine SVP model as a stage 2 palliation

151

model

152

A bidirectional cavopulmonary shunt (BCPS) was constructed by connecting the IVC

153

to the LA and the SVC to the main PA with two prosthetic conduits (Figure 1B). After154

the IVC-to-LA anastomosis was performed as described above, the SVC and PA were155

connected to the ePTFE graft under partial clamping of the PA with azygos vein

156

ligation. When all the anastomoses were completed, both the SVC and IVC were157

ligated at the right atrial junction.

158
159

Total cavopulmonary connection in a swine SVP model as a stage 3 palliation

160

model

161

A prosthetic conduit with two branches was constructed to deliver total venous

162

return directly into the PA as a total cavopulmonary connection (TCPC). This procedure163

was completed by graft anastomosis of the SVC to the PA, and another graft was164

anastomosed between the IVC and the graft in a T-configuration (Figure 1C).

165
166

Hemodynamic measurements and angiography

167

Hemodynamic measurements in the swine models were performed during intubation

168

when hemodynamics stabilized after model generation.

169

Hemodynamic variables in pigs were directly measured by placing a 22-gauge needle

170

connected to a pressure transducer (TruWave disposable pressure transducer;

171

Edwards Lifesciences, Irvine, CA, USA) into the location of interest. The total cardiac

172

output and systemic blood flow were measured using a pressure–volume system

173(ADV500 PV System, Transonic System, NY, USA). Vascular resistance was174

calculated using the following formula: Systemic ventricular resistance = (mean arterial

175

pressure-mean IVC pressure)/cardiac output*79.92 (dyne*sec/cm5). In this formula, the

8

176

IVC pressure was adopted for the precardiac pressure instead of the right atrial

177

pressure. Pulmonary vascular resistance was calculated as (mean pulmonary artery

178

pressure-mean LA pressure)/cardiac output*79.92 (dyne*sec/cm5). Selective

179

angiography was performed to check the postoperative blood flow.

180
181

Preparation of cardiosphere-derived cells (CDCs)

182

Piglet CDCs were prepared as described by Sano et al.5

183

Piglet right atrial tissues were minced and treated with 0.4% type II collagenase and184

0.01% DNase Ⅰ. Small atrial tissues were cultured to grow cardiospheres in

185

Dulbecco’s modified Eagle medium and F12 medium supplemented with 10% fetal

186

bovine serum and 2% penicillin/streptomycin.

187

CDCs were labeled with superparamagnetic iron oxide (SPIO) (Dragon green®

188

Bangs laboratories, Inc.). CDCs were cultured in SPIO added medium (30 microliter

189

Dragon green/10 ml medium) over night.

190
191

Cell therapy

192

CDCs were prepared for transplantation and verified as previously described.5, 6

193

Cell therapy was performed using the following three techniques:1) Intracoronary

194

CDC infusion was performed during temporary occlusion of the aorta, SVC, and IVC

195

via the aortic route. Both the SVC and IVC were then declamped to wash out the CDCs

196

into the coronary artery. After achieving steady-state cardiac contraction, the aorta was197

declamped. 2) Intraventricular infusion of CDCs was performed directly into the left

198

ventricle (LV) without aortic clamping. 3) Intravenous delivery of the CDCs was

199

performed by direct injection from the IVC.

200

The dose of CDCs administered to the swine LV and each model was 300,000

201

cells/kg of body weight, which was equivalent to previous research.5 Preliminary results

9

202

in rats showed that similar delivery to the myocardium was expected when 10 times the203

number of CDCs for coronary injection was administered into the LV. Therefore, the204

intracoronary administration was 30,000 cells/kg.

205
206

Magnetic resonance imaging

207

Ex-vivo MRI experiments in pigs were performed using a 1.5-Tesla Signa EXCITE208

MRI Scanner System with an 8-channel head array coil (GE Healthcare, Chicago, IL,

209USA) to assess cell distribution ex vivo. Two hours after CDC administration with an

210

artificial respirator and their chest left open, the pigs’ hearts were excised and211

transferred to the MRI suite and T2* weighted images of the extracted heart were212

acquired.6 The following settings were applied for T2* weighted imaging: repetition time

213(TR), 500 ms; echo time (TE), 4.5 ms; field of view, 24 ×24 cm; slice thickness, 5 mm;

214

gap, 0 mm; data matrix, 512 × 512; and flip angle, 30°.

215
216

Semi-quantitative analysis of SPIO-labeled cells in MRI217

MRI data were analyzed using ImageJ software (Rasband, W.S., ImageJ, U.S.

218

National Institutes of Health, Bethesda, Maryland, USA). The DICOM images were219

converted to tagged image file format to analyze the data using the ImageJ software.

220

To calculate the ratio of the low-signal-intensity area to the total organ area, a low-221

intensity area was selected and measured semiautomatically. The low-intensity area

222

was measured in a representative slice of each organ and the ratio to the whole area of223

the organ was calculated.

224
225

Histological analysis

226

Myocardial sections were deparaffinized, dehydrated, stained with a Prussian Blue

227

Iron Stain Kit (Polysciences, Warrington, PA, USA), and counterstained with

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228

hematoxylin and eosin to detect intracellular SPIO distribution in the transplanted229

myocardium. The morphology and structure of the in-vivo–engrafted CDCs were230

observed in stacked images using a BZ-X700 Analyzer (Keyence, Osaka, Japan). The

231

number of Prussian blue-positive cells was calculated and corrected with those from232

the total cardiac tissues analyzed.

233
234

Statistical analysis

235

Data are expressed as mean ± SD. Normal distribution was tested using the Shapiro–

236

Wilk test. Multiple comparisons of MRI, histological analysis data, and hemodynamic

237

parameters among the study groups were performed using one-way analysis of238

variance, followed by Tukey’s post-hoc correction that adjusted for multiplicity in

239

comparing multiple same-size groups that had a normal distribution and standard

240

deviation. Survival after a specified cardiac procedure was compared using log-rank

241

tests to assess the distribution of time to death, which may differ among the surgical242

procedures. Statistical analyses were conducted using the SPSS software (version 27,

243

IBM, Armonk, NY, USA). Statistical significance was set at P<0.05.

244

Desirable sample size was calculated with G*Power3.1.9.7

245(https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-246

arbeitspsychologie/gpower.html,Heinrich-Heine-Universität, Düsseldorf).

247
248

RESULTS

249

Stage-specific swine models of SVP

250

To study CDC homing and engraftment in large animals, we developed three distinct

251

palliative stage surgical models with single-ventricle circulation in pigs (Figure 1A-C,

252

Video Abstract). The results of the physiological function study are summarized and253

compared with those of the BV controls (Table 1). The impact of markedly decreased

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254

SaO2 in the BCPS group (BCPS:48.8 ± 4.6% vs. BV:99 ± 0.8%, MBT:80.4 ± 6.5 %,255

TCPC:99.2 ± 0.8%, P<0.01) was further characterized by significantly lower oxygen

256

saturation in the PA (SpaO2) after the surgical procedure compared with the control,

257

MBT, and TCPC groups. The MBT group had an increased proportion of blood flow258

directed to the pulmonary circulation through the aortopulmonary shunt, leading to259

augmented SpaO2 compared with the BCPS and TCPC groups (P<0.01). In contrast to260

the MBT and TCPC groups, significant decrease in hemodynamic oxygen in the BCPS

261

group resulted in a reduced survival time (P<0.05; Table 1).

262
263

Bypassing the pulmonary circulation during intravenous injection of CDCs

264

augmented cell retention in the systemic ventricle

265

First, we examined the delivery route for CDC engraftment in BV porcine hearts. After

266

intracoronary injection of 9.0 ×105 verified CDCs, SPIO-labeled cells were readily

267

detectable with MRI and were distributed throughout the entire ventricle (Figure 2A).

268

Intraventricular delivery of 9.0×106 CDCs showed hypointense spots in ventricular269

muscles, whereas intravenous infusion of the same dose of CDCs revealed few

270

engraftments on the BV hearts (intracoronary: 48.4±8.1%; intraventricle: 47.9±7.7%;

271

intravenous: 1.6±1.1%; P<0.001; Figure 2A). To determine whether shunt types affect272

CDC homing in the single ventricle after intravenous administration by avoiding stem273

cell trafficking to the lungs, SPIO-labeled 9.0×106 CDCs were transplanted into pigs

274with three different palliative stages of a single-ventricle and compared with BV control

275

hearts.8 As shown in Figure 2A, a significant increase in the area of hypointense276

regions was found in MBT and BCPS groups compared with control. In contrast, the277

TCPC group exhibited low level of SPIO-labeled CDCs on MRI, which was comparable278

to those in BV controls (BV: 1.6±1.1%; MBT: 53.2±9.1%; BCPS: 45.0±9.0%; TCPC:279

1.2±0.6%; P<0.01). Histological sections of the heart were stained with Prussian blue

12

280

to expose the transplanted CDCs present on the myocardium. Histological examination

281

showed that the number of SPIO-labeled CDCs was consistent with the MRI results282(BV: 0.6±0.6 cells/mm2; MBT: 4.0±1.8 cells/mm2; BCPS: 3.4±1.3 cells/mm2; TCPC:

283

0.4±0.4 cells/mm2; P<0.01, Figures 2B and C).

284
285

Statistical power and sample size

286Power was calculated from the results obtained to be 0.91 for the low signal intensity

287

area in MRI and 0.70 for the histologically detected SPIO-labeled CDCs number. In

288

addition, from the effect size calculated based on the experimental data, the desired

289

sample size at a significance level of 0.05% was calculated to be 24 animals for MRI

290

data and 36 animals for the histological data.

291
292
293

DISCUSSION

294

Stem cell transplantation has emerged as an adjunctive therapy to enhance staged

295

surgical repair in patients with congenital heart disease. 5,7,9,10

296

However, cell-based experimental studies in animal models with SVP have never

297

been described because of the technical difficulties in reconstituting univentricular298

circulation in normally developed postnatal hearts.1,2 This is the first study to develop

299

three distinct stage single-ventricle models in pigs: MBT, BCPA, and TCPC. Our main300

finding was that the intravenously administered CDCs were well distributed in the MBT

301

and BCPS models.

302
303
304
305

Single-ventricle swine model
Creating a chronic, large animal model of SVP remains a significant challenge. Early
studies of univentricular circulation have substantially relied on acute experimental

13

306

models with systemic-to-pulmonary shunt procedures, which have been shown to

307

induce volume overload.11 In our experiments, a Y-shaped conduit was used to directly308

deliver blood flow from both the SVC and IVC into the LA to complete the right heart309

bypass. The advantage of this novel large animal model is the avoidance of

310

intracardiac surgery requiring cardiopulmonary bypass to achieve right heart exclusion-

311

like physiology. While the PA was not ligated, the coronary venous return was drained

312

into the pulmonary blood flow, in which high coronary sinus pressure had a negligible313

effect on LV dysfunction.12314

The hemodynamics of our swine MBT model were similar to those previously

315

reported.13 Despite the short procedure time, clamping of the brachiocephalic artery in

316

pigs for anastomosis of the artificial graft is controversial. For the anastomosis of a317

systemic pulmonary shunt, the left subclavian artery may be preferable to avoid brain318

ischemia.

319

An IVC-to-LA shunt was applied to our BCPS model to achieve partial right heart320

bypass. Although the experimental animals in our study died within 20 h after the321

surgical procedure, the cardiac output of the LV was maintained by an increased322

preload, unlike in previous studies results14,15, both the PA pressure and systemic

323

afterload remained unchanged with a moderate increase in pulmonary vascular

324

resistance.

325

Experimental Fontan circulation was initially described as the intravenous delivery of a326

Y-shaped conduit constructed using polyethylene cannulas, without the use of327

cardiopulmonary bypass.16 In our TCPC model, extracardiac conduit anastomoses

328

were performed to directly connect both the SVC and IVC to PA. However, all three329

models exhibited very short lifetimes. The causes of short longevity in these models

330

seem to differ. For the MBT model, volume overload to the LV was the main reason; for331

the BCPS model, low pulmonary blood flow and desaturation were critical; and for the

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TCPC model, intravascular volume loss with ascites retention led to circulatory failure.

333

To control the pulmonary-to-systemic flow ratio, heart failure management, and infusion

334

therapy to maintain intravascular fluid seemed to be the keys to increase the models’

335

longevity.

336

The left azygos vein in pigs differs from that in humans, which functions as a systemic

337

vein to an atrial shunt. Therefore, total right heart bypass in the strict sense was338

incomplete in this study’s model. Intravenously injected stem cells were tracked in the339

presence of the left azygos vein. Ligating the left azygos vein without cardiopulmonary

340

bypass is technically difficult and carries the risk of bleeding. The BCPS model was341

considered the most affected by the presence of the left azygos vein. It is believed that342

a systemic venous-atrial shunt, which humans do not normally have, leads to lower

343

blood oxygen levels.

344
345

CDC dosage

346

The number of CDCs administered was based on previous studies, but if it was

347

exactly equivalent to that of a previous study, 5 it would have been better to increase348

the dose to the coronary arteries by 10 times (300,000 cells/kg) compared to this study.

349

However, the piglets weighed 5–10 times more than those used in previous human350

studies, making it impractical to culture sufficient cells. Therefore, the intravenous dose351

was set at 300,000/kg and the coronary artery dose was set at 30,000/kg. However,352

this did not undermine the purpose of tracking the destinations of the injected cells.

353
354

Impact of administration technique for cell therapy

355

Recent clinical and preclinical studies have shown that the delivery route of the cells356

may potentially affect ventricular function after infarction. However, many aspects

357

remain unclear, including the appropriate cell dose for injection and the efficient route

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358

of stem cell administration in patients with SVP.17 To our knowledge, this is the first359

preclinical study to demonstrate the differential cardiac homing dynamics of360

transplanted CDCs in experimental models of SVP.

361

Regarding the cardiac homing of stem cells, the inefficiency of intravenous injection362

was more obvious than that of intracoronary injection. However, the non-invasiveness363

of the administration method may enable repeated administration of stem cells and364

overcome this inefficiency. Furthermore, this delivery approach may offer several

365

advantages, including repeated cell infusion to elicit salubrious effects cooperatively

366

through paracrine/endocrine mechanisms of intrinsic and extracardiac origin.18 In our

367

MBT and BCPS models, intravenously delivered CDCs can be diverted into the368coronary arteries during initial circulation by bypassing the pulmonary circuit in a369

systemic blood flow-dependent manner to enhance acute cell retention (Figure 3). As

370

anticipated, preferential engraftment in the lungs and other extra-cardiac organs may

371

subsequently occur during circulation. It is conceivable that endogenous

372

cardiomyocytes and stem/progenitor cells can be stimulated directly by transplanted

373

CDCs to proliferate and that anti-inflammatory responses and cell protection signaling374

may be provoked by the CDCs at the site of their systemic delivery.19

375
376

Limitations

377

Although this study was the first to develop chronic models of SVP, hemodynamic

378

instability after the surgical procedures limited this work to an acute study with a short379

survival period, which may restrict further studies to investigate stage-specific380

functional outcomes. The small sample size was a statistical limitation because there381

was insufficient power to demonstrate the anticipated effects.

382
383

Conclusions

16

384

Three-stage right heart bypass surgery was feasible with durable outcomes to study

385

early cardiac homing of the transplanted CDCs with hemodynamic variables

386

compatible with the single LV. Additionally, CDC delivery through the lung bypass

387

circuit is effective in targeting a single ventricle before TCPC completion.

388
389

Acknowledgement390

The authors wish to thank members in Intervention Technical Center in Kobe, Japan391

for their assistance in performing animal operation.

392
393

Author contributions

394

Takuya Goto: Conceptualization; Data curation; Investigation; Writing—original draft.

395

Daiki Ousaka Conceptualization; Data curation; Investigation; Kenta Hirai: Data

396

curation; Yasuhiro Kotani: Supervision; Writing—review & editing. Shingo Kasahara:

397

Supervision; Writing—review & editing.

398
399

Funding

400

This work was supported by grants from the Research Project for Practical Application

401

of Regenerative Medicine (l8bk0104052h0003) by the Japan Agency for Medical

402

Research and Development.

403
404

Conflict of interest

405

None of the authors has any conflicts of interest to disclose.

406
407

Data availability statements

408

The data underlying this article will be shared on reasonable request to the409

corresponding author.

17

410

FIGURE LEGENDS:

411

Central image

412

Intravenously injected stem cells in stage-specific pig models were detected in the413

myocardium of stage 1 and 2 palliation models.

18

414

Figure 1.

415

Stage-specific swine models of single-ventricle physiology (SVP). The experimental

416

models of single ventricular circulation with modified Blalock–Taussig shunt (MBT) (A),417

bidirectional cavopulmonary shunt (BCPS) (B), and total cavopulmonary connection

418(TCPC) (C) and their angiograms are shown.

419

19

420

Figure 2.

421(A) Early cardiac homing after CDC injection in MRI. Among the intravenously infused422

groups, MBT and BCPS had significantly larger hypointensity areas than that in the423

normal biventricular group or TCPC group after CDC administration (*; versus BV;

424

P<0.01, versus TCPC; P<0.01, #; versus BV; P<0.05, versus TCPC; P<0.05). Labeled

425

CDCs were intravenously administered to the BV, MBT, BCPS, and TCPC models, and426

the CDC trafficking to the heart was assessed. (B) Histological analysis was performed427

to verify the cell identity by Prussian blue staining in each experimental model. Scale428

bar: 50 µm. (C) SPIO-labeled cells were counted and corrected by the area of total

429

heart section analyzed.

430
431

20

432

Figure 3.

433

CDC trafficking to the heart in swine models of single ventricular circulation. In MBT

434

and BCPS models, intravenously injected CDCs may entirely fill into the left ventricle

435(LV) and circulate into a systemic blood flow including coronary artery before supplying

436

pulmonary blood flow (A and B). In the TCPC model, a subset of CDCs may circulate

437

into the pulmonary blood stream through a prosthetic conduit and most of them are438

trapped by the lungs before entering systemic circulation (C). In MBT and BCPS

439

models, early CDC dispersion in the lungs could be avoided by right heart bypass

440

during the first circulation after intravenous CDC infusion.

441

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442

Table 1. Hemodynamic measurement in swine models of single ventricle.
BV (n=5)

MBT (n=5)

BCPS (n=5)

TCPC (n=5)

P

HR (bpm)

81.2 ± 8.2

83.8 ± 5.8

73.0 ± 3.1

77.4 ± 9.3

0.13

Mean ABP (mmHg)

65.4 ± 2.7

50 ± 6.7*

54.7 ± 5.0

62.4 ± 5.2<0.01

Systolic ABP (mmHg)

85.6 ± 7.3

73.4 ± 13.6

77.8 ± 7.5

81.8 ± 5.9

0.21

Diastolic ABP (mmHg)

55.8 ± 1.8

38.4 ± 3.8*

43.2 ± 4.0*

52.8 ± 6.4<0.01

Mean PAP (mmHg)

13.2 ± 0.5

21.8 ± 2.4

12.2 ±1.314.8 ± 0.8<0.01

LAP (mmHg)

5.4 ± 1.5

7.6 ± 1.1

8.6 ± 0.5

4.6 ± 1.1

<0.01

SVCP (mmHg)

4.4 ± 0.5

4.2 ± 1.6

12.2 ± 1.3†

13.8 ± 0.4†

<0.01

IVCP (mmHg)

4.8 ± 0.8

5.2 ± 0.8

5.0 ± 1.6

14.8 ± 0.8‡

<0.01

1851.7 ±1913.4 ±1760.4 ±2127.1 ±

264.4

457.8

180.2

199.2

236.7 ±

345.3 ±

458.8 ±

456.4 ±PVR (dyne*sec*cm-5)

42.1

33.4

153.0

65.7

<0.01

CO (L/min)

2.7 ± 0.27

4.4 ± 0.14

2.3 ± 0.15

1.8 ± 0 .1

<0.01

SV (mL)31.4 ± 4.5

53.2 ± 2.5

31.0 ± 0.77

23.4 ± 3.2<0.01

EDV (mL)

67.9 ± 5.8

111.1 ± 4.5

58.8 ± 2.8

46.2 ± 4.4<0.01

ESV (mL)

36.5 ± 4.0

57.9 ± 6.6

27.8 ± 2.4

23.0 ± 1.4<0.01

EF (%)

46.3 ± 4.5

48.0 ± 3.8

52.7 ± 1.9

50.3 ± 2.6<0.01

SaO2 (%)

99 ± 0.880.4 ± 6.5$

48.8 ± 4.6||

99.2 ± 0.8<0.01

SpaO2 (%)

68.2 ± 6.0

75 ± 4.6$

32.4 ± 3.5||

61.6 ± 5.3<0.01

Op time (min)NA

123.6 ± 10.9#

94.6 ± 13.4

92 ± 12.1<0.01

Survival (hrs)NA

9.4 ± 5.0

1.8 ± 0.8**

9.8 ± 5.6

0.02

SVR (dyne*sec*cm-5)

<0.01

443

ABP, arterial blood pressure (mmHg); BCPS, bidirectional cavopulmonary shunt; BV,

444

biventricle; CO, cardiac output; EDV, end-diastolic volume; EF, ejection fraction; ESV,445

end-systolic volume; MBT, modified Blalock-Taussig shunt; HR, heart rate; IVCP,

446

inferior vena cava pressure; LAP, left atrial pressure; NA, not applicable; OP,447

operation; P, P value; PAP, pulmonary artery pressure; PVR, pulmonary vascular

448

resistance; TCPC, total cavopulmonary shunt; SV, stroke volume; SVCP, superior vena449

cava pressure; SVR, systemic vascular resistance (dyn・sec/cm5). Data are expressed450

as mean ± SD. *, P<0.05 vs BV and TCPC groups. †, P<0.01 vs BV and MBT groups.

451‡, P<0.01 vs BV, MBT, and BCPS groups. $, P<0.05 vs BV, BCPS, and TCPC groups.

452||, P<0.01 vs BV, MBT, and TCPC groups. #, P<0.05 vs BCPS and TCPC groups. **,453

P<0.05 vs MBT and TCPC groups.

454

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