Stem cell homing, tracking and therapeutic efficiency evaluation for stroke treatment using nanoparticles: A systematic review

Table 1 Molecular imaging modalities

Image type	Technique	Physical principle	Tracrer	In vitro imaging	Preclincal imaging	Clinical Imaging	Spatial Resolution	Temporal resolution	Penetration depth	Sensitivity	Strengths	Limitations
Optical imaging	BLI	Visible light	Luminescent proteins	Yes	Yes	No	3-5 mm	Seconds to minutes	1-2 cm	High (+++)	High sensitivity, non-radioactive, cell expansion	Low penetration depth, non-translational
	FLI	Visible or NIRF light	Proteins or fluorescent dyes	Yes	Yes	No	2-3 mm	Seconds to minutes	< 1 cm	High (++)	High sensitivity, non-radioactive	Low penetration depth, autofluorescence
Nuclear imaging	PET	High-energy γ-rays	Radioisotopes [89 Zr (78.4 h), 18 F (1.83 h), 11 C (0.34 h), 64 Cu (12.7 h), 68 GA (1.13 h)]	No	Yes	Yes	1-2 mm	Seconds to minutes	Limitless	High (++)	High penetration depth, high sensitivity	Radiation exposure, high cost
	SPECT	Low-energy γ-rays	Radioisotopes [99 mTc (6.03 h), 123 I (13.2 h), 111 In (67.4 h)]	No	Yes	Yes	1-2 mm	Minutes	Limitless	High (++)	High penetration depth, high sensitivity	Radiation exposure, high cost
Magnetic imaging	MRI	Radio waves	Contrast agents	No	Yes	Yes	0.02-0.1 mm	Minutes to hours	Limitless	Low	High penetration depth, non-radioactive, high spatial resolution	High cost, low sensitivity and contrast

PET: Positron emission tomography; SPECT: Single-photon emission computed tomography; BLI: Bioluminescence; FLI: Fluorescence; MRI: Magnetic resonance imaging; NIRF: Near-infrared fluorescence.

Table 2 Characteristics of the studies and the stem cells used

Ref.	Yr	Country	Cell type	Source of cells	Medium culture - %FBS
Lim et al[33]	2019	South Korea	MSC	Human (adipose tissue)	DMEM - 10%FBS
Wang et al[29]	2019	China	MSC	Human (umbilical cord)	EBM-2 - 0%FBS
Yun et al[30]	2018	South Korea	NSC	Human (telencephalon)	NR
Argibay et al[38]	2017	Spain	MSC	Rat (bone marrow)	IMDM - 10%FBS
Duan et al[37]	2017	China	MSC	Rat (bone marrow)	DMEM - 10%FBS
Lu et al[35]	2017	China	NPC-Imm	Mice (C17.2)	DMEM - 10%FBS
Zhang et al[34]	2017	China	NSC	Rat (lateral ventricles)	StemPro NSC - 0%FBS
Lin et al[36]	2017	China	MSC	Rat (bone marrow)	DMEM -10%FBS
Zhang et al[39]	2016	China	NSC	Human (bone marrow)	NR
Duan et al[40]	2016	China	MSC	Rat (bone marrow)	DMEM - 10%FBS
Bai et al[42]	2015	China	MSC	Mice (bone marrow)	EBM-2
Chen et al[28]	2015	China	MSC	Human (umbilical cord)	DMEM-HG
Tan et al[41]	2015	Japan	MSC	Rat(bone marrow)	DMEM - 10%FBS,
Janowski et al[44]	2014	Poland	NSC	Human (umbilical cord)	DMEM-F12 - 2%FBS
Park et al[43]	2014	South Korea	MSC	Rat	DMEM - 0%FBS
Zhang et al[45]	2013	China	NPC-Imm	Mice (neonatal cerebellum)	DMEM - 10%FBS
Tarulli et al[46]	2013	Canada	MSC	Rat (bone marrow)	αMEM - 20%FBS
Liu et al[47]	2013	China	NSC	Rat (neonate)	DMEM-F12
Wang et al[48]	2011	China	MSC	Mice (bone marrow)	DMEM
Lee et al[50]	2009	Singapore	MSC	Human (fetal bone marrow)	DMEM - 10%FBS
Song et al[49]	2009	South Korea	NPC-Imm	Human (HB1.F3)	DMEM - 5%FBS
Kim et al[51]	2008	South Korea	MSC	Human	DMEM - 0%FBS
Guzman et al[52]	2007	United States	NSC	Human	HNCM
Syková et al[53]	2006	Czech Republic	MSC, rOEC	Mice; Human; Rat	NR
Zhu et al[54]	2005	United States	NSC	Human	NR

MSC: Mesenchymal stem cells; NSC: Neural stem cells; NPC-Imm: Neural progenitor cell - immortalised; ESC: Embryonic stem cell; rOEC: Rat olfactory ensheathing cells; C17.2: An immortalised mouse neural progenitor cell line; HB1.F3: An immortalized, clonal human NSC line; DMEM: Dulbecco's modified Eagle medium; DMEM-HG: Dulbecco's modified Eagle's medium high glucose; DMEM-F12: 50:50 mixture of DMEM and Ham's F12 medium; αMEM: Minimum essential medium Eagle: Alpha modification; EBM-2: Endothelial cell growth basal medium; IMDM: Iscove's modified Dulbecco's medium; FBS: Fetal bovine serum; StemPro NSC: Human neural stem cell culture medium; HNCN: Human neurosphere culture medium; NR: No reported.

Table 3 Characteristics of the contrast agents used in the stem cell labeling, homing and tracking analysis by molecular imaging modalities

Ref.	Contrast agent	Concentration (mg/mL)	Core/Hydrodynamic size (nm)	Coating agent	Zeta Potential (mV)	Conjugated agent (Ex/Em: nm)	Image detection mode	R1/R2 (mmol-1. Sec-1)	Developer
Lim et al[33]	NP (BCN-Fe3O4)	NR	20/238.9	BCN, chitosan	+12.6	Cy5.5 (675/695)	Dual (Mgt, NIRF)	NR/526.1	Synthesized
Wang et al[29]	Alkyl-SPIO	NR	NR/80-120	Alkyl-PEI	Approximately +21.0	NA	Dual (Mgt, BLI)	NR/549.7	Synthesized
Yun et al[30]	Zn0.4Fe2.6O4 (ZnMNP)12	NR	NR	Dextran	NR	NA	Dual (Mgt, BLI)	NR	Synthesized
Argibay et al[38]	Fe3O41	NR	3.7/94	Dextran	-11.0	NA	Mono (Mgt)	NR/701	Synthesized
Duan et al[37]	Fe3O4-LCP	0.12	6/136	PDLLA	+18.0	NA	Mono (Mgt)	NR/500.2	Synthesized
Lu et al[35]	PAsp(DMA)-Lys-CA2 (C-NP)2	NR	NR/64.1	NR	+15.32	Nile red (552/636)	Dual (Mgt, VFL)	NR/460.5	Synthesized
	PEG-Lys-CA2 (N-NP)2		NR/69.4		+0.10			NR/462.9
Zhang et al[34]	Ferritin2	NA	NA	NA	NA	NA	Dual (Mgt, NIRF)	NR	Synthesized
Lin et al[36]	SPION	0.25	NR/128	ASP	+21.6	NA	Mono (Mgt)	NR/296	Synthesized
Zhang et al[39]	SPION	NR	30/50	Dextran	NR	NA	Mono (Mgt)	NR/300	Synthesized
Duan et al[40]	Fe3O4-LCP2	0.12	6/136	PDLLA	+18.0	NA	Mono (Mgt)	NR/500.2	Synthesized
	Fe3O42	1.00	6/10.8	PLL	+32.8			NR/457.2
Bai et al[42]	bCD-Gd	NR	NA/24.4	NA	NR	Cy5.5 (675/695) Rhod (565/620)	Tri (Mgt, NIRF, VFL)	8.6/NR	Synthesized
Chen et al[28]	GRMNB1	NR	NA/130	Silica	NR	NA	Dual (Mgt, BLI)	1.21/127.89	Synthesized
Tan et al[41]	γ-Fe2O3 (ferucarbotran)	27.90	4/60	Carboxydextran	NR	NA	Mono (Mgt)	NR	Resovist®, Fujifilm RI Pharma Co. Ltd., Tokyo, Japan
Janowski et al[44]	Fe3O4	2.00	8/35	Dextran	+31.0	Rhod (565/620)	Dual (Mgt, VFL)	30.4/75.8	BioPAL Inc, Worcester, MA, USA
Park et al[43]	PCION	NR	11/371.6	PEG	+28.6	Rhod (565/620)	Dual (Mgt, VFL)	NR	Synthesized
Zhang et al[45]	fmSiO4@SPIONs	NR	30/151 30/148	Silica	-22.5	FITC (490/525)	Dual (Mgt, VFL)	NR/309.53	Synthesized
								NR/231.74
	fdSiO4@SPIONs				-38.0
Tarulli et al[46]	Fe3O4 (MPIO)	NR	NR/900	DBP	< 0	DGF (480/520)	Dual (Mgt, VFL)	NR	NR
Liu et al[47]	SPION	NR	NR	NR	NR	NR	Mono (Mgt)	NR	NR
Wang et al[48]	Fe3O4 (PMNC)	NR	8/120	Silica	-38.0	Rhod (565/620)	Dual (Mgt, VFL)	3.81/435	Synthesized
Lee et al[50]	MGIO	NR	5/602	PMG	NR	NA	Mono (Mgt)	NR	Synthesized
Song et al[49]	FeO1.44 (Feridex)	11.20	5-6/50-180	Dextran	-12.0	NA	Mono (Mgt)	23.9/98.3	Advanced Magnetic, Cambridge, MA, United States
Kim et al[51]	FeO1.44 (Feridex)	11.2	5-6/50-180	Dextran	-12	NA	Mono (Mgt)	23.9/98.3	Advanced Magnetic, Cambridge, MA, United States
Guzman et al[52]	FeO1.44 (Feridex)	11.2	5-6/50-180	Dextran	-12	NA	Mono (Mgt)	23.9/98.3	Berlex Laboratories, Wayne, NJ, United States
Syková et al[53]	Fe3O4 (Endorem)	15.8	4.3-5.6/150	Dextran	-12	NA	Mono (Mgt)	40/160	Guerbet, Roissy, France
Zhu et al[54]	FeO1.44 (Feridex)	11.2	5-6/50-180	Dextran	-12	NA	Mono (Mgt)	23.9/98.3	Advanced Magnetic, Cambridge, MA, United States

¹transduced with the luciferase protein (Vector Type - Lentiviral FUGW-Luc2).

²transduced with the GFP protein (Vector type - eGPF/FTH). Ex/Em: Excitation/Emission; NP: Nanoparticle; BCN: Bicyclo[6.1.0]nonyne; Fe₃O₄: Iron oxide; SPIO: Superparamagnetic iron oxide; LCP: Loaded cationic polymersomes; ZnMNP: Zinc-doped ferrite magnetic nanoparticle; PAsp(DMA): Poly(aspartic acid-dimethylethanediamine); Lys-CA: Lysine-cholic acid; C-NP: Cationic nanoparticle; PEG: Polyethylene glycol; N-NP: Neutral nanoparticle; SPION: Superparamagnetic iron oxide nanoparticles; bCD-Gd: Bacterial cytosine deaminase-gadolinium; GRMNBs: Gold nanorods crystal-seeded magnetic mesoporous silica nanobeads; PCION: Poly-(ethylene glycol)-coated cross-linked iron oxide nanoparticles; fmSiO₄@SPIONs: Fluorescent mesoporous silica-coated SPIONs; fdSiO₄@SPIONs: Fluorescent dense silica-coated SPIONs; PMNC: Polystyrene magnetite nanocluster; MGIO: Microgel iron oxide; MPIO: Micron-sized superparamagnetic iron oxide particles; NR: Not reported; NA: Not applicable; Alkyl-PEI: Amphiphilic low molecular weight polyethylenimine; PDLLA: Poly(D,L-lactide); PEI: Polyetherimide; ASP: Spermine-modified amylose; PLL: Poly-L-lysine; DBP: Divinyl benzene polymer; PMG: Precursor microgel; Cy5.5: Cyanine5.5; siRNA: Small interfering RNA; Rhod: Rhodamine B; pDNA: Plasmid DNA; FTH: Ferritin heavy chain; FITC: Fluorescein isothiocyanate; DGF: Dragon green fluorophore; Mgt: Magnetic; NIRF: Near infrared fluorescence; BLI: Bioluminescence imaging; VFL: Visible field fluorescence.

Table 4 Stem cell labeling process

Ref.	Cells	Passage	Contrast agent	Concen-tration μg/mL)	Incu-bation time (h)	Strategy of interna-lization	Effici-ency	Quantification			Cellular viability		Others analysis
								(pgFe/cell)	Technique		Method	Results
Lim et al[33]	MSC	P5-P7	NP(BCN-Fe3O4)	300	2	Ac4ManNAz	98.7%	15.3	ICP-MS		CCK-8 assay	> 95%	CF, SEM, CEM, MRI
Wang et al[29]	MSC	P2-P7	Alkyl-SPIO	an appropriate amount of Alkyl-PEI/SPIO (N/P = 20)	6	PEI	High Eff.	NA	NA		CCK-8 assay	> 90%	BLI, MRI
Yun et al[30]	NSC	NR	ZnMNP	50	NR	PLL: 1.5 g/mL	NR	4.6	NR		NA	NA	TEM, BLI
Argibay et al[38]	MSC	P0-P2, P9, P17	Fe3O4	100	24	PLL: 1.5 μg/mL	High Eff.	0.9-7.7	ICP-OES		LDH assay	NSD	TEM
Duan et al[37]	MSC	P3-P5	Fe3O4-LCP	15	1.5	PEI	Approximately 100%	Approximately 9	AAS		CCK-8 assay	> 90%	TEM, MRI
Lu et al[35]	NPC	NR	C-NP	10	4	PLL and Lipofectamin	Approximately 99.3%	NA	NA		CCK-8 assay	> 95%	MRI, VFL
			N-NP				Approximately 8.7%
Zhang et al[34]	NSC	P2-P3	Ferritin	MOI: 10	24	PLL	Approximately 63%	3.5	AAS		CCK-8 assay	NSD	TEM, MRI, PB
Lin et al[36]	MSC	P5-P9	ASP-SPION	30	1	NA	Approximately 100%	2.68	AAS		CCK-8 assay	> 90%	MRI, TEM
Zhang et al[39]	NSC	NR	Anti-CD15-SPION	NR	NR	NA	NR	NA	NA		NA	NA	NA
Duan et al[40]	MSC	P3-P5	Fe3O4-LCP	15	1.5	PEI	LCP > PLL	8.373	AAS		CCK-8 assay	> 90%	TEM, in vitro MRI
			Fe3O4	25	24	PLL		9.214
Bai et al[42]	MSC	NR	bCD-Gd	2 μmol	24	PLL	NR	NA	NA		NA	NA	NA
Chen et al[28]	MSC	NR	GRMNB	10	2	NR	High Eff.	33.62	AAS		MTT	87.6	BLI
Tan et al[41]	MSC	NR	Ferucarbotran	NR	24	NA	Approximately 95%	NA	NA		NA	NA	NA
Janowski et al[44]	NSC	NR	Fe3O4	25	48	PLL: 375 ng/mL	NR	NA	NA		NI	NI	NA
Park et al[43]	MSC	NR	PCION	1	0.25	PLL, EMF	NR	NA	NA		NA	NA	TEM, MRI
Zhang et al[45]	NPC	NA	fmNP	5, 10, 20, 33	0.5, 1, 2, 3	NA	fmNP > fdNP	5-30	ICP-AES		CCK-8 assay	90%-98%	TB
			fdNP					1-2.5			NA	NA	NA
Tarulli et al[46]	MSC	NR	MPIO	18.8	24	NA	95%	54	Flow cytometry		Flow cytometry	Approximately 94%	CF
Liu et al[47]	NSC	NR	SPION	14	24	NA	NR	NR	NR		NI	NI	NA
Wang et al[48]	MSC	NR	PMNC	0.5 mmol	1	NA	Approximately 100%	16-20	ICP-OES		CCK-8 assay	> 95%	TEM, CF, MRI
Lee et al[50]	MSC	P5, P6	MGIO	50	24	NA	Approximately 97%	33.3	ICP-OES		NI	> 95%	TEM, microarray
Song et al[49]	NPC	NR	Feridex	112.4	72	NA	Approximately 100%	0.2	AAS		TB	Unaffected	MRI
Kim et al[51]	MSC	NR	Feridex	1	12-16	PS	NR	2.6	ICP/MS		NI	NI	NA
Guzman et al[52]	NSC	NR	Feridex	5	24	PS: 2.5 μg/mL	98%	Halved every 3 d (%)	Semiquantitative (MRI)		Cell counting	Approximately 92%	NA
Syková et al[53]	MSC, rOEC	NR	Endorem	112.4	48-72	NA	NR	NA		NA	NA	NA	TEM
Zhu et al[54]	NSC	NR	Feridex	NR	1	Effectene	High Eff.	NA		NA	NR	NI	NA

MSC: Mesenchymal stem cells; NSC: Neural stem cells; ESC: Embryonic stem cell; rOEC: Rat olfactory ensheathing cells; P: Passage; NR: No reported; NA: Not applicable; NP: Nanoparticle; BCN: Bicyclo[6.1.0]nonyne; Fe₃O₄: Magnetite; SPIO: Superparamagnetic iron oxide; ZnMNPs: Zinc-doped ferrite magnetic nanoparticles; LCP: Loaded cationic polymersomes; C-NP: Cationic nanoparticle; N-NP: Neutral nanoparticle; ASP: Spermine-modified amylose; SPION: Superparamagnetic iron oxide nanoparticle; bCD-Gd: Bacterial cytosine deaminase-gadolinium; GRMNB: Gold nanorods crystal-seeded magnetic mesoporous silica nanobeads; MOI: Multiplicities of infection; PCION: Poly-(ethylene glycol)-coated cross-linked iron oxide nanoparticles; fmNP: FmSiO4@SPIONs; fdNP: FdSiO4@SPIONs; MPIO: Micron-sized superparamagnetic iron oxide particles; PMNC: Polystyrene magnetite nanocluster; MGIO: Microgel iron oxide; Alkyl-PEI: Amphiphilic low molecular weight polyethylenimine; MOI: Multiplicities of infection; Ac4ManNAz: Tetraacetylated N-azidoacetyl-D-mannosamine; PEI: Polyethylenimine; PLL: Poly-L-Lysine; EMF: External magnetic field; PS: Protamine sulfate; High Eff.: Hight efficiency; LCP: Loaded cationic polymersomes; AAS: Atomic absorption spectrophotometer; CCK-8: Cell counting kit-8; LDH: Lactate dehydrogenase; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TB: Turnbull blue; NSD: No significant differences; CF: Confocal fluorescence; SEM: Scanning electron microscope; CEM: Cryoelectron microscope; MRI: Magnetic resonance image; BLI: Bioluminescence image; TEM: Transmission electron microscopy; VFL: Visible field fluorescence; PB: Prussian blue.

Table 5 Stroke models induced by filament intraluminal middle cerebral artery, brain injury evaluation and animal features

Ref.	Ischemia mechanism	Animals					n / N	Ischemia type	Ischemia time (min)	Filament type	Anesthesia	Brain induction area (AP; ML to bregma in mm)	Blood flow analysis	Injury evaluation
		Specie	Type	Sex	Weight (g)	Age (wk)
Yun et al[30]	MCAo	Rat	SD	M	240-260	Adult1	3-8/50	T	30	3-0 nylon suture	NR	MNI	NI	TTC, MRI
Argibay et al[38]	MCAo	Rat	Wistar	M	280-300	NR	6/133	T	45	silicon rubber-	3%-4% sevoflurane	MNI	Laser-Doppler	MRI
Duan et al[37]	MCAo	Rat	SD	M	250-280	Adult1	6/54	NR	NR	NR	NR	MNI	NI	MRI
Lu et al[35]	MCAo	Rat	SD	NR	NR	NR	6/12	T	90	4-0 nylon suture, silicone coated tip	1% halothane	MNI	NI	MRI
Zhang et al[34]	MCAo	Rat	SD	NR	250-280	Adult1	NR/30	T	120	NR	PB (40 mg/kg)	MNI	NI	MRI
Lin et al[36]	MCAo	Rat	SD	M	250-280	Adult1	6/18	T	120	NR	NR	MNI	NI	MRI
Zhang et al[39]	MCAo	Mice	C57BL/6J	NR	20-25	8	NR/45	T	20	Nylon poly-1-lysinecoated	PB (6 mL/kg)	MNI	NI	MRI
Duan et al[40]	MCAo	Rat	SD	M	NR	Adult1	NR/24	NR	NR	NR	NR	MNI	NI	MRI
Chen et al[28]	MCAo	Mice	C57BL/6J	M	25-30	Adult1	NR/NR	T	120	square knot using a 10 suture	CH (0.4 g/kg)	zygoma/squamosal bone	NI	MRI
Tan et al[41]	Lacunar infarction	Rat	Wistar	M	240-260	NR	NR/22	P	NA	NA	2%-4% ISO	0; 3	NI	MRI
Zhang et al[45]	MCAo	Mice	CD1	F	NR	4	NR/NR	P	NA	6-0 rounded tip nylon	NR	MNI	NI	MRI
Park et al[43]	MCAo	Rat	SD	NR	250-280	NR	8/16	T	120	Micro clip 24 mm	Rompum (10 mg/kg) + Zoletil (30 mg/kg)	MNI	NI	MRI
Liu et al[47]	MCAo	Rat	SD	M	160-180	NR	6-8/48	NR	NR	Nylon	10% CH (300 mg/kg)	MNI	NI	MRI
Wang et al[48]	MCAo	Mice	CD1	F	NR	4	7/21	T	180	6-0 rounded tip nylon	NR	NI	NI	MRI
Song et al[49]	MCAo	Rat	SD	M	250-300	NR	3/6	T	120	NR	4% ISO	MNI	NI	MRI
Kim et al[51]	MCAo	Rat	SD	M	250-300	NR	2-6/13	P	NA	NR	ket. (80-100 mg/kg) + AM (5 mg/kg)	MNI	EEG	MRI
Guzman et al[52]	MCAo	Rat	SD	M	NR	Adult1	5/10	P	NA	NA	ISO	MNI + rhinal fissure	NI	MRI
	Global	Mice	NOD-SCID	NR	NR	0-1PN	12-16/28	T	5-10	NA	Cryoanestrhetized	NA	NI	MRI

¹Adult: Rat with 8-16 wk and mice with 6 to 20 wk. MCAo: Middle cerebral artery occlusion; SD: Sprague-Dawley; CD1: An outbred mice derived from a group of outbred Swiss mice; NOD/SCID: Nonobese diabetic/severe combined immunodeficiency; M: Male; F: Female; NR: No reported; n/N: Number of animals per group/total number of animals; T: Transient; P: Permanent; PN: Postnatal; NA: Not applicable; Ket: Ketamine; Xyl.: Xylamine; ISO: Isoflurane; AM: Aceprozazine maleate; CH: Chloral hydrate; PB: Pentobarbital; MNI: Midline neck incision; EEG: Electroencephalogram; TTC: Triphenyltetrazolium chloride; MRI: Magnetic resonance imaging.

Table 6 Stroke models induced by the photothrombosis of middle cerebral artery, brain injury evaluation and animal features

Ref.	Ischemia mechanism	Animals					n / N	Photosensitizer - rose bengal (dose; via)	Laser application parameters				Anesthesia	Brain induction area(AP; ML to Bregma in mm)	Injury evaluation
		Specie	Type	Sex	Weight(g)	Age (wk)			Time (min)	Diameter (mm)	Wavelengh (nm)	Power (W)
Lim et al[33]	PT	Mice	Balb/c nude	M	20-25	10	3-5/19	10 mg/mL; penile vein	16	NR	561	NR	Zoletil (50-30 mg/kg i.p.)	0.5; 2.5	MRI, NIRF, TTC
Wang et al[29]	PT	Mice	Balb/c nude	F	20-23	8	4-6/39	100 mg/kg	15	4	NR	NR	PB (50 mg/kg i.p)	-2.0; 2.0	MRI
Bai et al[42]	PT	Mice	Db/Db	M	NR	8	4/8	100 mg/kg; i.p.	15	NR	NR	NR	1% ISO	0.0; 2.0	MRI, NIRF
		Mice	Wild type	M	NR	8	10/20	100 mg/kg; i.p.	15	NR	NR	NR	1% ISO	0.0; 2.0	MRI, NIRF
Tarulli et al[46]	Focal devascularization	Rat	Long Evans	M	NR	8-12	3/9	NA	NA	NA	NA	NA	ISO + Ketoprofen	3.0/-4.0; 1.5/4.5	MRI
Lee et al[50]	PT	Rat	Wistar	F	NR	NR	NR/22	7.5 mg/mL; tail vein	10	3	603	60	Ket. (7.5 mg/100 g) + Xyl. (1 mg/100 g)	-2.0; -3.0	MRI, TTC
Syková et al[53]	Photochemical	Rat	Wistar	M	NR	8-12	NR/NR	NR	NA	NA	NA	NA	NR	NI	MRI

Blood flow analysis was not reported in any of the selected studies that used stroke models induced by photothrombosis; due to the model induction, all studies showed permanent ischemia after occlusion induction in the specific brain region. n/N: Number of animals per group/total number of animals; W: Watts; AP: Anterior-posterior; ML: Medial-lateral; PT: Photothrombosis; Db/Db: Diabetic mice model; M: Male; F: Female; NR: No reported; i.p.: Intraperitoneal; NA: Not applicable; ISO: Isoflurane; PB: Pentobarbital; Ket: Ketamine; Xyl.: Xylamine; MRI: Magnetic resonance imaging; NIRF: Near-infrared fluorescence; TTC: Triphenyltetrazolium chloride.

Table 7 Magnetic resonance imaging features for stem cell homing evaluation

Ref.	Equipment system	Analysis software	MF (Tesla)	Sequence	Weighted images (TR/TE; ms)	FOV; MT; ST (mm)	Homing evaluation time
Lim et al[33]	PC - Agilent Technologies	ImageJ (NIH)	9.4	T2	T2: 4000/32.5	NA; NA; 1.0	1, 3, 7, 10, 14 d
Wang et al[29]	PC - PharmaScan - Bruker	ImageJ (NIH)	7.0	TSE FLASH GRE	T2: 3000/NA	20 × 20; 256 × 256; 1.0	1, 3, 7 d
		ParaVision (Bruker)			T2*: 159.4/5	55 × 55; 256 × 256; 1.0
Yun et al[30]	Philips Medical Systems; an animal coil	NA	3.0	FSE	T2: 4000/80	50; 256 × 256; 0.5	1 d, 3 w
Argibay et al[38]	PC - Bio Spec - Bruker; surface coil array	ImageJ (NIH)	9.4	MGE	T2*: 2.9/1.5	19.2 × 19.2; 192 × 192; 1.0	4 h
Duan et al[37]	Achieva - Philips Medical Systems; 4-channel rat coil	ImageJ (NIH)	3.0	FSE	T2: 800/60	60; 256 × 256; 1.0	1-4, 6-8 wk
				FFE	T2*: 500/18
Lu et al[35]	Achieva - Philips Medical Systems; 4-channel rat coil	NA	3.0	FSE	T2: 200/31	60 × 60; 267 × 268; 1.0	1, 3, 7, 14 d
				FFE	T2*: 500/18
Zhang et al[34]	Achieva - Philips Medical Systems	ImageJ (NIH)	3.0	FSE	T2: 800/60	60 × 60; 256 × 256; 1.0	1-6 wk
				PDW	PDW: 3000/20
				FFE	T2*: 500/18
Lin et al[36]	Intera - Philips Medical Systems	ImageJ (NIH)	3.0	Multi SE	T2: 2000/20-80	80 × 80; 160 × 266; 2.0	1-6 wk
Zhang et al[39]	PC - PharmaScan - Bruker	ImageJ (NIH)	7.0	Turbo RARE	T2: 6000/ 60	30; 256 × 256; 0.5	2 d, 8 d
				FLASH GRE	T2*: 400/3.5
Duan et al[40]	Achieva - Philips Medical Systems	ImageJ (NIH)	3.0	FSE	T2: 800/60	60; 256 × 256; 1.0	1, 2, 3, 4, 6 wk
				PDW	PDW: 3000/20
				FFE	T2*: 500/18
Bai et al[42]	PC - PharmaScan - Bruker	ImageJ (NIH)	7.0	SE	T1: 500/15	20 × 20; 256 × 256; 1.0	1, 3, 5, 7, 10, 14 d
				FSE	T2: 2000/50
Chen et al[28]	PC - Bio Spec - Bruker	ImageJ (NIH)	7.0	RARE SE	T2: 3000/50	25.6; 256 × 256; 0.7	3 d, 7 d, 14 d
Tan et al[41]	PC - Unity INOVA, Varian	NR	7.0	SE	T2: 2500/60	30 × 30; 512 × 512; NR	1-42 d
Janowski et al[44]	Sonata Maestro Class - Siemens; 8-channel head coil	Osirix (Pixmeo) Amira (Visage Imaging)	1.5	SWI	T2*: 49/40	230; 168 × 256; 1.6	1 d, 1 wk, 1 mo, 2 mo, 4 mo
Park et al[43]	Achieva - Philips Medical Systems; animal coil	NA	3.0	SE	T2: 11000/125	NA; 284 × 286; 0.7	0 h, 2 d
Zhang et al[45]	Sigma - GE Healthcare; a human head coil	NA	3.0	FSE	T2: 5840/104	45 × 45; 256 × 256; 1.5-2.0	1 d, 3 d
				Map MSME SE	T2: 3500/20-160
Tarulli et al[46]	Sigma - GE Healthcare	NA	3.0	FSE	T2: 4500/35-75	40 × 40 × 17; 256 × 256; 1.0	1 d, 7 d, 14 d
				3D-SPGR	T2*: 25/7	40 × 40 × 20; 256 × 256; 1.0
Liu et al[47]	Sigma - GE Healthcare; a rat coil		3.0	T2*	T2*: 2560/6.8	6.0; NR; 1.6	1, 7, 21 d
Wang et al[48]	Sigma - GE Healthcare	NA	3.0	FSE	T2: 5840/104	45 × 45; 256 × 256; 1.5	1, 7, 30 d
Lee et al[50]	Sigma - GE Healthcare; a clinical coil	NA	1.5	TSE	T2: 2000/81	90; 192 × 192; 1.5	0, 1, 5, 12 d
				GRE	280/20	20; 160 × 160; 1.5
Song et al[49]	Sigma - GE Healthcare	NA	1.5	T2	T2: 3500/80	60 × 60; 256 × 160; 2.0	1d, 3d, 1-4 wk
				3D GRE	T2*: 50/20	80 × 80; 256 × 160; 2.0
Kim et al[51]	PC - Bio Spec - Bruker	NA	4.7	SE	T1: 600/14	40 × 30; 256 × 192; 1.0	2 d, 1 w, 2 w...10 wk
				RARE	T2: 5000/90
				FLASH	T2*: 758 × 30
Guzman et al[52]	PC - Varian Medical Systems	NA	4.7	SE	T2: 2500/45	40; 256 × 256; 1.0	2 d, 7 d, 35 d
				3D GRE	T2*: 600/5	30 × 30 × 30; 128× 128 × 128
Syková et al[53]	PC - Bio Spec-Bruker	NA	4.7	FGE	T2: NA	NA	1 d, 1-7 wk
Zhu et al[54]	Sigma - GE Healthcare	NA	3.0	SE	T2: 200/20	NA	1 d, 7 d

MRI: Magnetic resonance imaging; PC: Preclinical MRI scanner; NIH: National Institutes of Health; NA: Not applicable; MF: Magnetic field; T2: Transverse relaxation time; FSE/TSE: Fast or turbo spin echo; FFE: Fast field echo; PDW: Proton density-weighted; GRE: Gradient echo; MGE: Multiple gradient echo; SPGR: Spoiled gradient recalled echo; SE: Spin echo; FGE: Fast gradient echo; FLASH: Fast low angle shot; PDW: Proton density-weighted; RARE: Rapid acquisition with refocused echoes; SWI: Susceptibility weighted imaging; MSME: Multi-spin-multi-echo; SPGR: Spoiled gradient recalled echo; TR: Time repetition ; TE: Echo time ; FOV: Field-of-view; MT: Matrix; ST: Slice thickness.

Table 8 Near-infrared fluorescence imaging features for stem cell homing evaluation

Ref.	Agent	Equipment	Software	Excitation / Emission wavelength (nm)	Time of exposition	Follow-up
Lim et al[33]	DBCO-Cy5.5	IVIS Lumina Series III (PerkinElmer)	Living Image (PerkinElmer)	670/NA	1 min	In vivo at 1, 3, 7, 10, 14 d; ex vivo at 2, 27, 30, 33, 36 h
Zhang et al[34]	LV-FTH-EGFP	small animal in vivo FLI system (in vivo FxPro; Carestream)	MI (Carestream)	487/509	NA	1, 2, 3, 4, 5, 6 wk
Bai et al[42]	Cy5-5	Maestro in vivo imaging system (CRi, Woburn)	Maestro v. 2.10.0	675/695	NA	1, 3, 5, 7, 10, 14 d

DBCO: Dibenzylcyclooctyne; Cy5.5: Cyanine 5.5; LV-FTH-EGFP: Lentiviral vector-encoding ferritin heavy chain and enhanced green fluorescent protein; NA: Not applicable; MI: Molecular imaging software; Cri: Cambridge research and instrumentation.

Table 9 Bioluminescence imaging features for stem cell homing evaluation

Ref.	Lentiviral vector	Equipment	Software	Substrate	Dose	Image acquisition	Follow-up
Wang et al[29]	Luc2/eGFP	IVIS Lumina Series III (Perkin-Elmer)	NR	D-luciferin (Promega, United States)	100 mL (30 mg/mL)	10 min after injection	1 d, 3 d, 7 d
Yun et al[30]	Fluc/eGFP	IVIS® Spectrum imaging system (Perkin Elmer)	NR	D-luciferin (Promega, United States)	150 mg/kg	NR	1 d, 1 wk, 3 wk
Chen et al[28]	Luc/GFP	IVIS Imaging System 200 Series (Caliper)	Living Image 3.0 (Xenogen Corp.)	D-luciferin (Caliper)	270 mg/g	15 min after injection	0, 14 d

All substrates were administered intraperitoneally. Luc: Luciferase; Fluc: Firefly luciferase; GFP: Green fluorescent protein; eGFP: Enhanced GRP; NR: Not reported.

Table 10 Stem cell administration, homing and cellular therapeutic efficiency

Ref.	Cell Type	Immuno-genicity	Time from stroke (h)	Cell administration			Groups	Follow-up	Outcome
				Route	Number	Volume (µL)			Behavior	Infarct volume	Mole-cular proteins/others	Cells mi-gration
Lim et al[33]	MSC	XNG	NR	IC-CTL	1 × 106	5	Stroke + cells vs Stroke-cells	1, 3, 7, 10, 14 d	NR	(+)	NR	(+)
Wang et al[29]	MSC	XNG	24	ITC1	5 × 105	100	Alkyl-SPIO/siPHD2 > Alkyl-SPIO/si	1, 3, 7 d	(+) mNSS; FFT at 14 d	(+) 7 d	(+) Ki67; CD31 -7 d; (+) NeuN -14 d	(+)
	MSC	XNG	24	ITC1	5 × 105	100	Alkyl-SPIO/si vs saline	1, 3, 7 d	(+) mNSS; FFT at 14 d	(-) 7 d	(+) Ki67; CD31 -7 d; (+) NeuN -14 d	(+)
Yun et al[30]	NSC	XNG	24	IA-IC	3 × 106	100	Mag-Cells > UL-Cells/sa-line	0, 3, 5, 7, 21 d	(+) Cilinder at 21d	NR	(+) MAP2; Nestin; GFAP; TuJ1 -7d	(+)
Argibay et al[38]	MSC	ALG	8	IA; IV-jugular	2 × 105; 1 × 106	300	D-MNP-labeled MSC (IA × IV)	4, 24, 72 h	(-) Cilinder	(-) at 14d	(-) CD31; Ki67; DCX	(+)
Duan et al[37]	MSC	ALG	48	IC-CTL	5 × 105	3	Labeled cell > UL-cells	1, 2, 3, 4, 6, 8 wk	(-) mNSS	(-)	(-) TUNNEL (-) GFP	(+)
							Labeled/UL vs control	1, 2, 3, 4, 6, 8 wk	(+) mNSS at 3, 4, 6, 8 wk	(+) at 4, 6, 8 wk	(+) TUNNEL 7-21 d, (+) GFP 7-21 d	(+)
Lu et al[35]	NPC	ALG	NR	IC-IPS	5 × 105	2.5	labeling with N-NPS	0, 3, 7, 14 d	NR	(+)	(+) Nestin	(+) low
							labeling with C-NP	0, 3, 7, 14 d	NR	(+)	(+) Nestin	(+)
Zhang et al[34]	NSC	XNG	48	IC-CTL	5 × 105	3	FTH-EGFP-NSC > non trans-ducec NSC	1, 2, 3, 4, 5, 6 wk	(+) mNSS at 1-6 wk	(+) at 1-6 wk	(+) GFAP; Nestin; CD11b at 6 wk	(+)
Lin et al[36]	MSC	ALG	48	IC-CTL	5 × 105	NR	ASP-SPION vs UL vs PBS	1, 2, 3, 4, 5, 6 wk	(-) mNSS	(-)	(-) GFAP; NeuN; CD11	(+)
Zhang et al[39]	NSC	XNG	7d	IC-CTL	NR	7	Stroke pure > Stroke + Ara-C	0, 2, 8 d	NR	(+) at 8 d	(+) CD15+; Nestin at 8 d	(+)
Duan et al[40]	MSC	ALG	48	IC-CTL	5 × 105	3	PLL-SPION or PM > UL	1, 2, 3, 4, 5, 6 wk	NR	(+) at 4, 6 wk	(-) GFP	(+)
Bai et al[42]	MSC	XNG	24	IA - IC	1 × 106	100	DM + RWJ + cell > DM + cells	1, 3, 5, 7, 10, 14 d	NR	(+)	(+) p38 MAPK at 7 d	(+)2
Chen et al[28]	MSC	XNG	30 min	IV-femoral	5 × 105		Mag-cells > UL-cells	0, 3, 7, 14 d	(+) VM at 14, 28 d	(+) at 14 d	(+) TuJ1; NeuN; GFAP at 28 d; (+) RT-PCR1 at 28 d	(+)
Tan et al[41]	MSC	ALG	7 d	IC-CTL	5 × 105	10	Stroke + cells over time	0, 1, 7, 14, 21, 42 d	NR	NR	(-) GFP and NeuN at 7 d; (+) GFP and NeuN at 6 wk	(+)
Janowski et al[44]	NSC	AuTL	NR	IC	2 × 104	10	Case over time	0, 1, 7, 60, 120 d, 33 mo	NR	NR	NR	(+)
Park et al[43]	MSC	XNG	14d	IC-CTL	6 × 105	5	Pcion/pDNA MSC vs control	1, 2 d	NR	(-)	NR	(+)
Zhang et al[45]	NPC	ALG	24	IC-CTL	5 × 105	5	fsiSPION-NPC vs control	1, 3 d	NR	(+)	(+) Nestin	(+)2
	NPC	ALG	24	IV-tail	1 × 106	300	fsiSPION-NPC vs control	1, 3 d	NR	NR	(+) Nestin	(+)
Tarulli et al[46]	MSC	XNG	72	IV-tail	3 × 106	700	MPIO-BMSC vs UL-BMSC	1, 7, 14 d	NR	NR	NR	(+)
Liu et al[47]	NSC	XNG	NR	IC-CTL	3 × 104	5	Stroke + NSC_FA > Stroke + NSC	1, 7 d	NR	NR	(+) Sox-2 BrdU at 21 d	(+)
Wang et al[48]	MSC	XNG	7d	IC-CTL	1 × 105	5	FMNC-MSC > UL-MSC vs control (FMNC)	0, 1, 7, 30 d	NR	NR	(+) TuJ1	(+)
Lee et al[50]	MSC	XNG	48	IC-CTL	2 × 104	5	M600-MSC vs FC-MSC	1, 5, 12 d	NR	NR	NR	(+)
	MSC	XNG	48	IV-tail	2 × 106	500	M600-MSC vs control	5, 12 d	NR	NR	NR	(+)
Song et al[49]	NPC	XNG	24	IC-IPS	4 × 105	5	FO-NPC vs control	1, 3, 7, 14, 21, 28 d	NR	NR	(+) BrdU; GFAP at 28 d	(+)
	NPC	XNG	24	IV-tail	4 × 106	500	FO-NPC vs control	1, 3, 7, 14, 21, 28 d	NR	NR	(+) BrdU; GFAP at 28 d	(+)
Kim et al[51]	MSC	AuTL	7d	IC-IPS/CTL	1 × 105	2	Feridex®-labeled hMSC over time for both vias	2d, 1, 2, 4, 6, 8, 10 wk	NR	NR	(-) GFAP; TH; MAP2; TuJ1; Nestin at 10 wk	(+)
Guzman et al[52]	NSC	XNG	7d	IC-CTL	3 × 105/5 × 104		NSC-SCns-SPION	3, 9, 12,18 wk	NR	NR	(+) SC121 or SC101; TuJ1; GFAP; MAP2 at 18 wk	(+)
Syková et al[53]	rOEC	ALG	NR	IC-CTL	NR	NR	OEC-SPION over time	3-7 wk	NR	NR	(+) NeuN; GFAP at 28 d	(+)
	MSC	XNG	NR	IV-femoral	NR	NR	MSC over time	6-30 d	NR	NR	(+) NeuN; GFAP at 28 d	(+)
Zhu et al[54]	NSC	AuTL	NR	IC	NR	NR	Patients treat with NSC and no treat	2 yr	(+) SEP and DRS at 6, 9 mo	(+) cells uptake by PET at 3, 6 mo	NI	(+)

¹left ventricle.

²In addition to cell migration analysis, studies reported biodistribution analysis after stem cell administration. The Bay study[42] reported biodistribution in the liver, spleen, heart, lungs, and kidneys; Zhang et al[45] reported that the SPION-labelled cells IV > IA at 3 d after injection were detected in spleen, liver, heart, kidney, and lung. MSC: Mesenchymal stem cells; NSC: Neural stem cells; ESC: Embryonic stem cell; rOEC: Rat olfactory ensheathing cells; NR: No reported; XNG: Xenogeneic; ALG: Allogeneic; AuTL: Autologous; IC: Intracerebral; IC-CTL: IC contralateral; ITC: Intracardially; IC-IPS: IC ipsilateral; IV: intravenous; IA-IC: Intraarterial through internal carotid artery; Alkyl-SPIO: Amphiphilic low molecular weight superparamagnetic iron oxide; Mag: External magnet; UL: Unlabeled; siPHD2: siRNA against PHD2; C-NP: Cationic nanoparticle; N-NP: Neutral nanoparticle; FTH-eGFP: ferritin heavy chain: Enhanced green fluorescent protein; ASP-SPION: Spermine-modified amylose superparamagnetic iron oxide nanoparticle; FBS: Fetal bovine serum; Ara-C: Cytosine arabinosine; PLL: Poly-L-Lysine; PM: Polymersone; PCION: Poly-(ethylene glycol)-coated cross-linked iron oxide nanoparticles; MPIO: Micron-sized superparamagnetic iron oxide particles; DM: Diabetes mellitus; RWJ: RWJ67657; fsiSPION: fmSiO4@SPION; FC: Ferucarbotran; FA: Folic acid; FO: Ferumoxide; FMNC: Fluorescent-magnetite-nanocluster; mNSS: Modified neurological severity score; FFT: Foot-faults test; VM: Vertical movement; SEP: Somatosensory evoked potential; DRS: Disability rating scale; MAP2: Microtubule-associated protein 2; GFAP: Glial fibrillary acidic protein; TuJ1: Neuron-specific class III beta-tubulin; GFP: Green fluorescent protein; MAPK: Mitogen-activated protein kinase; RT-PCR: Reverse transcription polymerase chain reaction; BrdU: 5′-Bromo-2′-deoxyuridine; TH: Tyrosine hydroxylase.