Supplementary Materials and Methods

Cell culture

Human iPSCs (409B2) were provided by the RIKEN BRC through the

National Bio-Resource Project of the MEXT, Japan. hiPSCs were cultured with

mTeSR1 (veritas) and iMatrix (Nippi) coated vessels without feeder cells. For

assessment of iNCMSCs, 409B2-hiPSCs were used as positive control to detect

some PSCs specific markers.

KG-1, acute myeloid leukemia cell lines, was provided by the National

Institutes of Biomedical Innovation, Health and Nutrition through JCBR Cell

Bank, Japan. KG-1 was cultured in 20%FBS-IMDM (gibco), and used for positive

control of hematopoietic stem cell markers.

Human bone marrow derived MSCs were purchased from LONZA

(Catalog # PT-2501, Lot # 0000451491), and cultured in same condition to

iNCMSCs. In brief, the medium was 10%FBS-aMEM supplemented with 5 ng/mL

bFGF, and passaged with 0.25% Trypsin-EDTA. The cells from passage 3 were

used for all assays.

Supplemental Table 1

FACS antibodies

Target (antigen)

Conjugate Company Catalog No.

CD271 PE BD 557196CD44 FITC Biolegend 338804CD73 PE Biolegend 344004CD90 FITC Biolegend 328108

CD105 APC Biolegend 323208CD34 FITC BD 555821CD45 PE Biolegend 304007

TRA-1-60 Alexa Fluor 488 Biolegend 330614

rBC2LCN FITC Wako 180-02991

Supplemental table 2

IHC antibodiesTargetAntigen Company Catalog

No. Antigen Retrieval 1st Abdilution

1st Abreaction

COL1 abcam ab6308 Protainase K, 5min 1/ 100 RT, 1hrCOL2 Kyowa Pharma Chemical F- 57 Protainase K, 5min 1/ 500 RT, 1hrCOL10 eBioscience 14- 9771 Protainase K, 5min 1/ 500 RT, 1hrSOX9 Santa Cruz Biotech sc- 20095 (- ) 1/ 100 4℃, O/ N

hVimentin abcam ab16700 (- ) 1/ 100 RT, 1hrKi- 67 DAKO M7240 citrate buffer (pH6.0), 95℃, 10min 1/ 100 4℃, O/ N

Supplemental table 3

List of TaqMan gene expression assays

Gene symbol assay code threshholdGAPDH Hs02758991_g1 0.5POU5F1 Hs00999632_g1 0.1NANOG Hs04260366_g1 0.1Lin28a Hs00702808_s1 0.1SOX2 Hs00602736_s1 0.1p16 Hs00923894_m1 0.5

TFAP2A Hs01029413_m1 0.5Nestin Hs04187831_g1 0.5Sox10 Hs00366918_m1 0.1Pax3 Hs00240950_m1 0.1

CD271 Hs00609976_m1 0.5CD140a, PDGFRa Hs00998026_m1 0.5CD140b, PDGFRb Hs00387364_m1 0.5

CD44 Hs01075861_m1 0.5CD73, NT5E Hs00159686_m1 0.5

CD34 Hs02576480_m1 0.5CD45 Hs04189704_m1 0.1

COL1A2 Hs00164099_m1 0.1COL2A1 Hs00264051_m1 0.1COL10A1 Hs00166657_m1 0.1

ACAN Hs00153936_m1 0.1SOX9 Hs01001343_g1 0.1SOX5 Hs00753050_s1 0.5SOX6 Hs00264525_m1 0.5Osterix Hs01866874_s1 0.5

Osteopontin Hs00959010_m1 0.5RUNX2 Hs01047973_m1 0.5

Addiponectin Hs00605917_m1 0.5LPL Hs00173425_m1 0.5aP2 Hs01086177_m1 0.5

Supplementary Figure legends

Fig.S1 NC/MSC marker analysis for long passaged iNCMSCs (P4, P8)

(A): FACS analysis of neural crest and mesenchymal stem cells surface markers

in iNCMSCs (P4, P8).

(B): Positive controls of CD34 and CD45 using KG-1 which is acute myeloid

leukemia cell lines.

Abbreviation: iNCMSCs, induced neural crest derived mesenchymal stem cells;

BM-MSCs, bone marrow derived mesenchymal stem cells.

Fig.S2 iNCMSCs expanded without bFGF sustained CD90 expression.

(A): Cell morphology of iNCMSCs at P4 expanded with or without bFGF. Scale

bars = 100 μm.

(B): FACS analysis of CD90 of iNCMSCs at P4 expanded with or without bFGF.

(C): CD90 positive cell rate during expansion with or without bFGF. Each symbol

represents each passage.

Abbreviation: bFGF, basic fibroblast growth factor.

Fig.S3 The properties of human BM-MSCs in vitro.

(A): Cell morphology of human BM-MSCs passaged twice with 10%FBS-aMEM, 5

ng/mL bFGF.

(B): Oil red staining of human BM-MSCs cultured in adipogenesis medium at day

9.

(C,D): Osteogenesis of human BM-MSCs cultured in osteogenesis medium. ALP

staining at day 7 (C) and Alizarin Red S staining at day 14 (D).

(E): Saf-O staining of chondrogenic pellet cultured with TGFb3 and BMP2 at day

28.

(F): The gross appearance of hBM-TEC developed in 24 well plate at day 7.

Scale bars = 100 μm (A-D) and 500 μm (E).

Fig.S4 Immunostaining for collagens.

(A-C): Immunostaining for COL1 and COL2 for each group at 1 month and 2

month.

Right panels were magnification image of black arrow of left panels. Scale bars

= 500 μm and 100 μm (high magnified images.)

Abbreviations: COL1, type 1 collagen; COL2, type 2 collagen; TEC, tissue

engineered construct; hBM, human bone marrow derived mesenchymal stem

cells; iNCMSC, induced neural crest derived mesenchymal stem cells.

Fig.S5 Tumorigenesis of iNCMSCs after in vivo transplantation.

(A): Histological analysis of iNCMSC-TEC transplanted knee at 2 month. HE

staining and immunostaining (hVimentin and Ki-67) were performed with serial

sections.

(B): EB-outgrowth cells derived from 253G1-iPSCs were transplanted into

osteochondral defect of nude rat. HE staining and Ki-67 immunostaining at 1

month specimen.

Scale bars = 500 μm (upper panels) and 100 μm (lower panels).

Abbreviation: iNCMSCs, induced neural crest derived mesenchymal stem cells;

EB, embryoid body; HE, hematoxylin and eosin.

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