Table 2.

A methodological pattern of studies on assessing the effects of cell-based products’ implantation into a defect of the vocal folds

	In vitro characterization	Cell type and detection method after IMP	Markers for MA	Histological staining	Markers for IHC	Morphometric parameters	In vivo assessment	Physical properties
								Mechanics	Vibration
Kanemaru et al. [49]	FACS (Sca-l, CD29, CD34, CD44, CD45, CD4ge, and 7-AAD)	Xeno (murine) BM-MSC GFP tracing	No	H&E	Keratin Desmin	No	No	No	No
Hertegard, et al. [23]	FACS (CD73, CD90, CD105, CD166, CD14, CD31, CD34, CD45, and CD80)	Xeno (human) BM-MSC FISH	No	H&E	Collagen type I	Collagen density	No	No	No
Lee et al. [45]	No	Auto AD-MSC CM-Dil tracking	No	H&E	No	No	Direct laryngoscopy	No	No
Johnson et al. 2010 [52]	– FACS (CD11b, CD19, CD-45, 7-AAD) – AlamarBlue assay – ICC (CD-44, Sca-1)	Xeno (murine) BM-MSC GFP tracing	COL3A1 FN1 HAS3 HYAL3 SMA TGF- β1	No	No	No	No	No	No
Xu et al. [46]	– Phase contrast microscopy – MTT assay – FACS (CD34, CD44, CD105, CD106) – ICC (Vimentin) – HS (Oil red O, Von Kossa) – SEM	Auto AD-MSC DAPI tracking	No	H&E MT AB	FN	Collagen, HA, FN density	Endoscopy	No	No
Ohno et al. [50]	No	Auto BM-MSC ND	No	vG AB	No	LP thickness Collagen and HA density	No	No	No
Kim et al. [60]	No	Xeno (murine) BM-MSC GFP tracing	COL1A1 HAS2 FN1 TGF- β1	H&E MT AB	Collagen type I	Collagen and GAG density	Endoscopy	No	No
Kim et al. [38]	– FACS (CD73, CD90, CD31, CD34, CD-45) – Differentiation test (AG, OG, CG)	Xeno (human) AD-MSC CM-DiI tracking	No	H&E MT VvG	HGF FSP-1 Collagen type I FN	Collagen, elastin, and FN density	Endoscopy	No	No
Peng et al. [27]	– FACS (CD29, CD44, CD90, CD105e, CD34, CD45) – Clonogenicity assay – Differentiation test (AG, OG, CG)	Allo EM-MSC Dil tracking IHC (SMA, vimentin)	No	H&E MT vG AB	FN	Relative content of collagen, elastin and HA	Comparativeendoscopy	No	No
Choi et al. [25]	No	Xeno BM-MSC In vivo DiD tracking	–	H&E MT AB PR		Scar index measurement Intensity of collagen optical density	Endoscopy	No	No
Hu et al. [39]	– Phase contrast microscopy – ICC (vimentin, collagen)	Auto AD-MSC/VF fibroblasts/dAD-MSC ND	No	No	Collagen Elastin HA Decorin FN	No	Endoscopy	No	No
Hiwatashi et al. [40]	No	Allo (murine) BM-MSC and AD-MSC GFP tracing	FGF2 HGF COL1A1 COL3A1 HAS1 HAS2 HAS2 MMP1 VEGFa	vG AB	No	LP thickness collagen and HA density	No	No	No
Svensson et al. [57]	No	Xeno (human) ESC FISH	No	H&E	Collagen type I	LP thickness Collagen density Scored fibrosis analysis	No	No	No
Valerie et al. [53]	No	Allo AD-MSC ND	No	H&E MT AB RO	No	LP thickness Scored collagen, elastin and HA density	No	No	No
De Bonnecaze et al. [41]	– FACS (CD34, CD45, CD31, CD73, CD90) – Differentiation test (AG, OG)	Auto AD-MSC GFP tracing via LV transduction	No	H&E PR O	Collagen type I FN	LP and EP thickness PR optical density Density of elastic fibers Number of inflammation foci (foci/mm3)	No	No	No
Shiba et al. [47]	– HA (H&E, vG, MT) – IHC (vimentin, panCK)	Auto AD-MSC in fibrin gel ND	No	H&E vG MT	No	No	Endoscopy	No	No
Imaizumi, et al. [26]	No	Xeno (human) iPSC FISH	No	H&E vG MT	panCK AFP ALPP	Collagen and elastin density Retention of hydrogel	No	No	No
Morisaki et al. [61]	FACS (CD29, CD90, CD45)	Allo AD-MSC GFP tracing	HAS1 HAS2 HAS3 COL1A1 COL3A1 MMP1 MMP8 FGF2 HGF VEGFa	vG AB	No	Collagen and HA density	No	No	No
Bartlett et al. [54]	No	Xeno (human) BM-MSC Human β-actin expression	COL1A2 COL3A1 FN1 FMOD LPL OCN HAS HYAL2 IL1β IL17β, TGF-β1, TNF INγ αSMA	MT VvG AB	No	Relative collagen, elastin and HA content	No	No	No
Goel et al. [48]	No	Allo AD-MSC FISH, PCR (Y CS) TUNEL labeling for apoptotic cells	No	H&E MT	No	No	No	No	No
Svistushkin et al. [62]	No	Auto BM-MSC ND	No	H&E PR O	Collagen type I and III	LP thickness Thickness of collagen fibrils Scored analysis of 11 morphological parameters Collagen type I/III ratio	No	No	No
Hertegård et al. [22]	– FACS (CD73, CD90, CD105, HLA-I, CD14, CD34, CD45, HLA-II) – Migration and invasion assay – qPCR (HAS, HYAL, MMP) – ELISA (IL8, HGF, IL6, VEGF, TGF-β1)	Xeno (human) MSC FISH	No	AB	Collagen type I	Fibrosis, inflammation, MPS intensity Scored fibrosis analysis	No	No	No

AB Alcian blue, AD-MSC adipose tissue-derived mesenchymal stromal cells, AFP alpha 1 fetoprotein, AG adipogenic, Allo allogeneic, ALPP placental alkaline phosphatase, Auto autological, BM-MSC bone marrow-derived mesenchymal stromal cells, CG chondrogenic, COL1A1 procollagen I type, COL1A2 procollagen alpha 2, COL3A1 procollagen III type, CS chromosome, dAD-MSC differentiated adipose tissue-derived mesenchymal stromal cells, EM epiglottis mucosa, EP epithelium, ESC embryonic stem cells, FGF2 fibroblast growth factor 2, FN1 fibronectin (MA), FMOD fibromodulin, FN fibronectin, FSP-1 fibroblast specific protein 1, GAG glycosaminoglycans, HA hyaluronic acid, HAS1 hyaluronan synthase-I, HAS2 hyaluronan synthase-II, HAS3 hyaluronan synthase-III, HGF hepatocyte growth factor, HLA human leucocyte antigen, HS histological staining, HYAL3 hyaluronidase 3, FACS fluorescence-activated cell sorter analysis, ICC immunocytochemical staining, IHC immunohistochemical staining, IL1β interleukin 1β, IL17β interleukin 17β, INγ interferon γ, iPSC induced pluripotent stem cells, LP lamina propria, LPL lipoprotein lipase, LV lentivirus, MA molecular analysis, MMP1 matrix metalloproteinase 1, MMP8 matrix metalloproteinase 8, MPS mucopolysaccharides, MT Masson trichrome, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, O orcein, OC osteocalcin, OG osteogenic, panCK pancytokeratin, PR picrosirius red, RO reticulin and orcein, SEM scanning electron microscopy, SMA smooth muscle actin, TGF-β1 transforming growth factor-beta 1, TNF tumor necrosis factor, VEGFa vascular endothelial growth factor type A, vG van Gieson, VvG Verhoeff-Van Gieson, Xeno xenogeneic