Supplementary Data s6

Supplementary Data

Materials and Methods

Microarray analysis

Based on the BeadArray technology CD34+ stem cells from UCB were analyzed with a

Sentrix HumanRef8 v2 Exoression BeadChip (Illumina, USA) and a selected marker profile.

Endodermal/hepatic and cell-cell communication related genes were determined after a cultivation period of 10 days. RNA isolation Illumina TotalPrep Amplification Kit was used concerning manufacturing instructions for RNA preparation. Generation of biotin labeld cRNA, hybridisation and Cy3 streptavidin labelling are performed concerning available protocols from Illumina.

Video imaging of co-culture

UCBC and AML12 cells were labeled as described in Material and Methods (Dye Transfer).

3x105 CalceinAM labelled AML12 cells were seeded out in a 24well plate. Co-culture was initiated after adhesion period of 24h by adding 1,5x105 pkH26 labelled UCBC. Co-culture was visualized by using an inverse microscope and AxioCamMR camera (Carl Zeiss

Microimaging) and analyzed with AxioCamMR software. Co-cultured was monitored by video imaging over 24 h using a time interval of 5 min. Tables

Table S1. Applied Biosystems TaqMan Gene Expression Assays® for real time

Assay ID Gene name Gene Tissue/ Specificity (Applied symbol Biosystem) Hs00173490_m1 AFP, hCG15197 AFP fetal liver, endodermspecific Hs00609411_m1 ALB, hCG14967 ALB adult liver, hepatocytes gap junction protein, tumorigenesis and Hs00702141_s1 GJB1, CMTX, CMTX1 Cx32 liver regeneration GJA1, AVSD3, gap junction protein, heart Hs00748445_s1 Cx43 DFNB38, GJAL development, cell-cell communication Hs00156373_m1 CD34, hCG21280 CD34+ human CD34+ adult stem cells Hs00269972_s1 C/EBPA, hCG20142 C/EBP adult liver, hepatocytes KRT8, CARD2, CK-8, epithelial tissue, cell differentiation, Hs01630795_s1 CK8 CK8, CYK8 structural integrity of cells KRT18, CYK18, K18, Hs01920599_gh CK18 early epithelial tissue, PIG46 structural integrity of epithelial cells, KRT19, CK19, K19, Hs00761767_s1 CK19 cell differentiation involved in K1CS embryonic placenta development intracellular communication, CDH1, cadherin 1, type Hs00170423_m1 E-Cadherin cell-cell adhesion (epithelial tissue), 1 tumorigenesis Hs00232764_m1 FOXA2 HNF3 fetal liver, definitive endoderm embryogenesis, myocardial Hs00171403_m1 GATA 4, ASD2, VSD1 GATA 4 differentiation and function, lineage determination Hs00230853_m1 HNF4A HNF4 adult liver, hepatocytes Hs01895061_u1 POU5F1 Oct-4 pluripotency Hs00751752_s1 SOX17 Sox17 fetal liver, definitive endoderm Hs99999905_m1 GAPDH, hCG2005673 GAPDH house keeping gene

The following cycling conditions were used for all of the above primer assays.

95°C 10 min.

Denaturation: 95°C 15 sec } 45 cycles Amplification: 60°C

Figures Figure S1. Video imaging of co-cultured AML12 and umbilical cord blood cells (CD34+ stem cells).

Over 24h co-culture of CalceinAM labelled murine AML12 (green) and pkH26 labelled

UCBC (red) was monitored by video imaging using a time interval of 5 min (20x magnification). The video demonstrated the rare and sporadic cell-cell contact between the two different cell types.