Development of MAPC derived induced endodermal progenitors Sambathkumar, Rangarajan

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University of Groningen

Development of MAPC derived induced endodermal progenitors Sambathkumar, Rangarajan

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2017

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Sambathkumar, R. (2017). Development of MAPC derived induced endodermal progenitors: Generation of pancreatic beta cells and hepatocytes. University of Groningen.

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90

Chapter 3

Human induced Endodermal Progenitors derivation & Characterization from Multipotent Adult Progenitor Cells

Rangarajan Sambathkumar, Renate Akkerman, Sumitava Dastidar, Philip Roelandt, Manoj Kumar, Ana Rita Mestre Rosa, Nicky Helsen, Veerle Vanslembrouck, Eric Kalo, Satish Khurana, Jos Laureys, Conny Gysemans, Marijke M Faas, Paul de Vos, and Catherine Verfaillie.

Article Under Revision

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Chapter 3 Human induced Endodermal Progenitors derivation & Characterization from Multipotent Adult Progenitor Cells

3.1 Abstract

New and renewable sources of cells are needed for cell based therapies of liver diseases and type 1 diabetes. Human pluripotent stem cells (PSCs) are one potential source to generate functional hepatocytes or β-‐cells. As an alternative, adult stem cells such as Multipotent Adult Progenitor Cells (MAPC) could be used. However, human MAPCs have less plasticity than PSCs, and their ability to generate endodermal cells is not robust. We hypothesized that hMAPCs could be reprogrammed to endodermal progenitor cells (induced endodermal progenitor cells, iENDO cells) that can be expanded long term and differentiated into both hepatocyte-‐ and endocrine pancreatic-‐like cells. We demonstrated that hMAPC were reprogrammed with 14 transcription factors (TFs) to an intermediate progenitor, the called induced endodermal progenitors (iENDO), with an epithelial morphology, that could be expanded long term and expressed endodermal genes. This iENDO cells could be used as source of cells to generate the hepatocytes and pancreatic endocrine β-‐cells.

3.2 Introduction

Generation of functional mature hepatocyte and insulin producing pancreatic endocrine β-‐cells for cell based therapy of liver failure and type 1 diabetes is a promising key area of regenerative medicine. Currently, the pharmaceutical industry is at an urgent need for reliable drug hepatotoxicity screening models, as drug-‐

induced liver injury is the most prevalent reason for drug withdrawal from the

market [1]. Hepatic cells, derived from stem cells could be a new source of cells for

hepatotoxicity screening. Liver transplantation for acute and chronic liver diseases

and transplantation of cadaveric pancreas or islets cells for diabetes treatments have

been used in the demand for an effective treatment [2-‐4]. However, they carry

several disadvantages such as scarcity of available donors, and immunorejection,

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Chapter 3 Introduction

92 categorized as embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs, which are pluripotent, are derived from the inner cell mass (ICM) of the blastocyst [5, 6].

ASCs can be derived from many postnatal tissues [7-‐10]. Human embryonic stem cells (ESC) and more recently induced pluripotent stem cells (iPSC) are cells, with the ability of self-‐renew and to differentiate into cells from the three germ layers [6, 11].

Mesendoderm and definitive endoderm cells are common precursor for the generation of mature endodermal cell types such as hepatocyte and insulin producing functional pancreatic β-‐cells [12]. Several protocols for In vitro differentiation to hepatocyte like cells (HLC) [13-‐17] and mature endocrine pancreatic β cells [18-‐20] from pluripotent stem cells have been developed including protocols by our group by mimicking in vivo liver and pancreatic β-‐ cells development. Nevertheless, to date, a hepatocyte differentiation protocol is not able to produce fully mature hepatocytes [21], but in the case of mature pancreatic β-‐ cells it is now possible with improved cell culture conditions [22, 23]. While ESCs have unlimited proliferation and differentiation potential, ASCs are more restricted.

However, the low risk for tumorigenesis associated with ASC make them a favorable choice. To extend the use of ASCs, many groups are evaluating whether it is possible to extend their tissue restricted differentiation ability.

Multipotent adult progenitors cells (MAPCs), a type of mesenchymal stem cell (MSC), possess broader differentiation characteristics than other adult stem cells. MAPCs have been isolated from rodent and human bone marrow (BM) [24, 25]. Rodent MAPC (renamed BM Hypoblast stem cells, HypoSC), derived from bone marrow following long-‐term culture [26], can differentiate into multiple cell types of the mesodermal and endodermal germ layer, including hepatocyte-‐like cells and insulin producing β-‐cell like cells [27, 28]. Unfortunately, human MAPCs (hMAPC) have less potency to be differentiated toward hepatocyte-‐like cells and insulin producing β-‐

cell like cells [26]. In addition, for hMAPC, we do not see a spontaneous reprogramming towards HypoSCs by extended culture.

To overcome this restricted potency of human MAPCs to differentiate into

hepatocytes and β-‐cells, we decided to use a new approach in which human MAPCs

were transdifferentiated to expandable endodermal progenitor cells (termed iENDO

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Chapter 3 Introduction

cells), which could than be differentiated to hepatocytes or β-‐cells. The rationale for the use of hMAPCs as starting population was threefold: (1) hMAPCs are derived from human bone marrow and can be expanded significantly (for ±70 PDs) without acquisition of genetic abnormalities; (2) hMAPCs (trade name MultiStem®) are currently being used clinically in the setting of ischemic disorders and as immunomodulators without known toxicity [29-‐31] (3) although hMAPCs can differentiate in vitro and in vivo in mesodermal cell types, including endothelium, they differentiate, unlike their rodent counterparts, less robust to endodermal cell types [16, 25, 28, 32].

The discovery of iPSCs and direct reprogramming of mouse and human fibroblasts to hepatocytes [33-‐37], and pancreatic β-‐cells by OSKM combined with small molecules approach or small molecule alone [38-‐40], has provided the basis for the hypothesis that the differentiation state, even of mature cells, can be manipulated. Therefore, investigators are testing whether it is possible to de-‐differentiate cells a few steps back, but not to a pluripotent state which is associated with teratoma formation.

This partial dedifferentiation could allow broader applications of MAPC. To overcome the restricted differentiation of hMAPCs towards hepatocytes and pancreatic β-‐cells, we here demonstrated that using a complement of transcription factors (TFs), chosen based on insights from early endoderm fate specification and differentiation, it might be possible to trans/de-‐differentiate hMAPC to an expandable induced endoderm progenitor (iENDO) state. In the following chapters, we will show that these cells could be differentiated towards hepatocyte and pancreatic β-‐cells in vitro and in vivo. In contrast to PSCs, such endodermal progenitors might represent a safer cell source, as differentiation to non-‐

endodermal cells would not occur. Compared to direct lineage conversion, such endodermal progenitors can be extensively expanded.

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Chapter 3 Materials and Methods

94 3.3 Materials and Methods:

3.3.1 Generation of lentiviral vectors carrying transcription factors

We selected 16 candidate TFs: ESC pluripotency factors (OCT4, SOX2, KLF4, CMYC), TFs expressed in early definitive endoderm (MIXL1, GATA4, SOX17, FOXA1, FOXA2, FOXD3, FOXF1), TFs expressed in late endoderm (HNF4α, HNF6, HNF1α, HEX, CEBPα). Primers were designed with including specific restriction enzyme sites in the flanking regions to amplify the cDNAs (supplementary table-‐1). The coding sequence for FOXA2, HNF1α, HNF4α, CEBPα, HNF6, and FOXA1, FOXF1, FOXD3 and for HEX, MIXL1, GATA4, SOX17 were PCR amplified from hESC differentiated to hepatocytes for 12 days, 6 days and 4 days, respectively and cloned into the PLVX-‐IRES-‐HYGRO constitutive CMV promoter based lentiviral vector plasmid purchased from (Cat No.

632185, Clontech, Cambridge, USA). Human OCT4, SOX2, KLF4 and CMYC cDNA were excised from pMXs-‐OCT3/4(Cat.No. 17217), pMXs-‐SOX2 (Cat No. 17218), pMXs-‐KLF4 (Cat No. 17219), pMXs-‐c-‐MYC (Cat No. 17220) respectively. The cDNAs were cloned into the PLVX lentiviral vector (Addgene, Cambridge, USA). Each TF construct was verified by colony PCR, restriction digestion pattern and cDNA sequence analysis.

Each TF containing lentiviral vector was co-‐transfected with 2nd generation lentiviral packaging (gag pol tat rev)/envelope (VSV-‐G) plasmids into Lenti-‐X™ 293T Cell line purchased (Cat No. 632180, Clontech, CA, USA) using Fugene transfection reagent (Cat No. E2311, Promega, Madison, WI, USA). Transfer vector-‐ 6μg, Packaging plasmid (psPAX2) (Cat No. 12260, Addgene, Cambridge, USA)-‐ 3.5μg, Envelope plasmid (pMD2. G) (Cat No. 12259 Addgene, Cambridge, USA)– 1.5μg. Supernatants containing the lentiviral particles were collected after 48hr, filtered through a 0.45μm filter and stored at -‐80

⁰

C for future use.

3.3.2 Culture conditions for BM-‐human MAPCs

Human Multipotent Adult Progenitors (hMAPCs) were derived from human bone

marrow as described in Roobrouck et al., [25]. MAPCs were cultured as previously

described[25].

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Chapter 3 Materials and Methods

3.3.3 Generation of induced endodermal progenitors (iENDO) from human MAPCs On day 0, 45,000 hMAPCs cells were plated in 10 cm

²

petri dishes (Cat No. 150350, Thermo Scientific™ Nunclon™ Delta treated, VWR International, Belgium) in triplicates. On day 1, cells were transduced with a cocktail of 14 or 16 un-‐

concentrated viral vector supernatants (MOI of 3). On day 4, transduced cells were harvested and replated on 1.8% differentiation Matrigel (Cat No. 356231, BD biosciences, Bedford, MA, USA) coated six well plates (Cat No. 3516, Corning-‐Costar

^®

, MA, USA). A part of the cells was used to evaluate transgene expression. From day 5 onwards, cells were maintained in endoderm induction medium containing 60%

DMEM low glucose (Cat No. 31885023, Gibco, Grand Island, NY, USA); 40% MCDB-‐

201 (Cat.No. M6770, Sigma-‐Aldrich, Saint Louis, MO, USA); 1x-‐Penicillin-‐

Streptomycin(10,000 U/mL) (Cat No. 15140122, Gibco, Carlsbad, CA, USA); 0.25x LA-‐

BSA (100x) (Cat No. L9530, Sigma-‐Aldrich, Saint Louis, MO, USA); 0.25x ITS-‐A (100x) (Cat No. 51300044, Gibco, Grand Island, NY, USA); 100nM,L-‐ascorbic Acid (Cat No.

A4403, Sigma-‐Aldrich, Saint Louis, MO, USA); 1μM dexamethasone (Cat.No. D2915, Sigma-‐Aldrich, Saint Louis, MO, USA), 50 μM, β-‐mercaptoethanol (50mM) (Cat No.

31350010, Gibco, Grand Island, NY, USA) supplemented with 100ng/ml Activin-‐A (Cat No. 120-‐14E), 50ng/ml Wnt3a (Cat No. 315-‐20) and 5ng/ml BMP4 (Cat No. 120-‐

05ET). All growth factors were purchased from Peprotech, USA. From day 9 to 15 morphological changes from mesenchymal to epithelial cells were assessed by bright field microscopy. Untransduced and PLVX-‐eGFP empty vector tranduced hMAPCs cultured under similar conditions were used as negative control. The iENDO cells were maintained in a 37

⁰

C, 21 % O

2

, 5 % CO

2

incubator. Between days 20-‐25 transcripts for endogenous mesendoderm/definitive endoderm and late endoderm marker genes were measured by qRT-‐PCR. Cells were fixed with 4%

paraformaldehyde (PFA) (Cat No. P6148, Sigma-‐Aldrich, Saint Louis, MO, USA) overnight at 4

⁰

C to perform immunostaining.

3.3.4 Expansion potential of iENDO cells

14TF iENDO cells were seeded at one million-‐cells/100 cm

²

petri dish (Cat No.

150350, Thermo Scientific™ Nunclon™ Delta treated, VWR International, Belgium)

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Chapter 3 Materials and Methods

96 coated with 0.1% gelatin. Afterwards every 4-‐5 days, cells were harvested with 0.25% Trypsin EDTA (Cat No. 25200056, Gibco, Grand island, NY, USA) and enumerated using a NUCLEOCOUNTER® NC-‐100™. Population doublings (PDs) were enumerated as the number of cells initially seeded (C

0

) to the number of cells harvested (C

1

) at each passage using the following equation: PDnew = PD initial + [log (C

1

/C

0

]/log2.

3.3.5 Maintenance and Expansion of hESCs

H9 hESCs (purchased from WiCell, Madison, WI, USA) were expanded on a 6 well plate (Cat.No. 3516, Corning-‐Costar

^®

, VWR International, Belgium), in feeder-‐free conditions on hESC-‐qualified BD Matrigel

^TM

, (Cat No. 354277, BD Biosciences, Bedford, MA, USA) using E8 medium Essential 8

^TM

Basal Medium Essential 8

^TM

Supplement (Cat. No. A1517001, Gibco, Grand Island, NY, USA).

3.3.6 RNA extraction, cDNA synthesis and gene expression

Total RNA was purified using the GenElute

^™

Mammalian Total RNA Miniprep Kit (Cat No. RTN350, Sigma-‐Aldrich, Saint Louis, MO, USA) and ZR RNA MicroPrep (Cat No.

R1061, Zymo Research, CA, USA). CDNA was generated using 0.5 -‐ 1μg of RNA with SuperScript® III First-‐Strand Synthesis SuperMix for qRT-‐PCR kit (Cat No. 11752050, Invitrogen, CA, USA) and qRT-‐PCR was performed on a ViiA™ 7 Real-‐Time PCR System with 384-‐well plate (Cat No. 4453536, Applied Biosystems, Carlsbad, CA, USA) with a Platinum® SYBR® Green qPCR SuperMix-‐UDG w/ROX (Cat No. 11744500, Invitrogen, CA, USA) and primers mix at final concentration of 250nM. Gene expression (Cycle threshold) values were normalized based on the PPIG (peptidylprolyl isomerase G) house keeping gene and the Delta CT calculated. Gene specific primers, purchased from IDT technologies, Leuven, Belgium were designed to distinguish between transgene (CDS-‐IRES) and endogenous (CDS-‐3’UTR or Exon-‐

Exon spanning) gene expression. Gene expression graphs or heat maps were

represented relative to the housekeeping gene PPIG in log scale. Heat maps were

generated using Gene E software (Broad Institute of MIT and Harvard, Cambridge,

MA, USA). The efficiency of primers was tested by serial dilution of cDNA and by

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Chapter 3 Materials and Methods

calculating coefficient of regression (R2). An efficiency of 95-‐105% with an R2≥ 97%

was considered as good (see supplementary table-‐2-‐4 for list of all qRT-‐PCR primers used in this study).

3.3.7 Immunostaining

Undifferentiated 14TF iENDO cells cultured on coverslips in 12 well plate were fixed with 4% (PFA) and permeabilised with 0.2% PBST, blocked with 0.2% PBST + 5%

normal donkey serum, and stained overnight at 4

^°

C with primary antibodies and respective isotype controls in Dako diluent. Slides were washed with 1xPBST three times. Immune complexes were detected by incubation with a Alexa-‐Fluro AF488 (Green) and AF555 (Red) (1:500) coupled to secondary antibodies for 30 min at room temperature. The nuclei were visualized using Hoechst or DAPI (1:2000). After 3 washes, slides were mounted with prolong Gold mounting medium. The signals were detected with a Nikon Eclipse Ti-‐S and Axioimager.Z1 microscope (Carl Zeiss).

Identical exposure times were used for isotype and specific antibodies. (See supplementary table 5-‐6, for list of primary, secondary, and isotype antibodies).

(Note: For cell surface protein CXCR4, stained with anti-‐human CXCR4 conjugated with PE antibody (1:100), cell permeabilisation and secondary antibody step was not followed).

3.3.8 Statistical analysis

Comparisons between two groups were analysed using an unpaired 2-‐tailed

Student’s t-‐test. P-‐values < 0.05(), < 0.01(), <0.001() were considered

significant. Data are shown as mean and error bars represent standard error of mean

(SEM) of minimum three independent experiments. All results were analyzed using

Graph Pad prism 6 software.

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Chapter 3 Results

98 3.4 Results:

3.4.1 14TFs can reprogram human MAPCs into induced endodermal progenitor (iENDO) cells

Initially, we transduced human MAPCs with 16 selected TFs, including the pluripotency TFs, OKSM and TFs known to be important in (mes) endoderm specification (Fig. 1A). Cells were maintained in endoderm differentiation medium with Activin-‐A/Wnt3a/BMP4 on Matrigel coated dishes from day 4 onwards (Fig. 1B).

On day 4, all TFs were highly expressed except for CEBPα (Fig. 1C). Between day 9 and 20, we observed morphological changes from a mesenchymal morphology to clusters of epithelial cells with a cuboidal cells in the transduced cells, but not in untransduced or PLVX-‐eGFP transduced cells (Fig. 1D).

A) Selected'16'Transcrip1on'factors'for'iENDO'induc1on' B)

Morphological observation of Mesenchymal to epithelial (MET) clusters derived from 16 TFs transduced BM-hMAPC

BM#hMAPC#Day+0+ BM#hMAPC+UTC#Day+20+

PLVX#empty#Day+20+ 16TFs+MAPC#Day+20+

100μm+

tOCT3AtSOX2tKLF4tCMYCtMIXL1tGATA4tSOX17tFOXA1tFOXA2tFOXD3tFOXF1tHNF4AtHNF6tHNF1AtHEX tCEBPA 10^-7

10^-6 10^-5 10^-4 10^-3 10^-2 10^-1 10⁰ 10¹

Relative expression to PPIG

Transgene expression

Untransduced hMAPC-Day 4 16TFs transduced BM-hMAPC-Day 4

* * * * *

*

* * * * * * * ^NS

NS

*

C)

E) F)

D)

G)

relative row minrow max

14TFs iENDO-Day 0 Day 28 iENDO-HLC.2D Day 28 hESC-HLC.2D PHH

Annotation ALB AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 Rela%ve'log' Row'min' Row'max'

Figure S1.16TF transduction in hMAPC create more hepatocyte like cells than iENDO

Pluripotency-Genes- ---Early-Endoderm- Late-endoderm- genes- OCT4,&SOX2,&KLF4&

CMYC,&FOXD3! MIXL1,&GATA4,&

SOX17,&FOXA1,&

FOXA2,&FOXF1!

HNF4A,&HNF6,&HEX,&

HNF1A,&CEBPA,&&!

16TFs2iENDO2Characteriza6on2at2day2202

relative row min row max

Annotation ALB AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4

Rela6ve2log2 Row2min2 Row2max2

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ hE SC $E nd od er m $D ay $4 $

SOX7$

GATA6$

eGATA4$

GSC$

eMIXL1$

EOMES$

FGF8$

eSOX17$

relative r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n SOX7

GATA6 eGATA4 GSC

eMIXL1 EOMES FGF8 eSOX17

Genes2

Mesendoderm2markers2

relative

r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n eFOXA2

eFOXA1 CER1 CXCR4 CKIT eHEX

ECADHERIN EPCAM

OCLN eFOXA2$

eFOXA1$

CER1$

CXCR4$

CKIT$

eHEX$

ECADHERIN$

EPCAM$

OCLN$

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $$D ay $2 0$ $ hE SC $E nd od er m $D ay $$4 $

Genes2

Deﬁni6ve2endoderm2/Epithelial2markers2

ALB$ $ AFP$ $ eHNF4A$

$ eHNF6$

$ PDX1$

relative row min row max

Untransduced hMAPC-Day 20 16TFs derived iENDO-Day 20 hESC diff mature endoderm cells -day 20

Annotation

ALB AFP

eHNF4A eHNF6 PDX1

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ $ hE SC $L ate $E nd od er m $D ay $2 0$

Genes2

Late2endoderm2markers2 A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Hepatocyte%markers%

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$ G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

*$

***$

**$

*$

***$

*$

***$

*$

***$

**$

***$

*$

***$

**$

*$

***$

**$

***$

**$

***$

**$

***$

**$

***$

**$

*$

***$

**$

*$

***$

16TFs2iENDO2Characteriza6on2at2day2202

relative row min row max

Annotation ALB AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ hE SC $E nd od er m $D ay $4 $

SOX7$

GATA6$

eGATA4$

GSC$

eMIXL1$

EOMES$

FGF8$

eSOX17$

relative r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n SOX7

GATA6 eGATA4 GSC

eMIXL1 EOMES FGF8 eSOX17

Genes2

relative

r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n eFOXA2

eFOXA1 CER1 CXCR4 CKIT eHEX

ECADHERIN EPCAM

OCLN eFOXA2$

eFOXA1$

CER1$

CXCR4$

CKIT$

eHEX$

ECADHERIN$

EPCAM$

OCLN$

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $$D ay $2 0$ $ hE SC $E nd od er m $D ay $$4 $

Genes2

ALB$ $ AFP$

$

eHNF4A$

$ eHNF6$

$ PDX1$

relative row min row max

Untransduced hMAPC-Day 20 16TFs derived iENDO-Day 20 hESC diff mature endoderm cells -day 20

Annotation

ALB AFP

eHNF4A eHNF6 PDX1

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ $ hE SC $L ate $E nd od er m $D ay $2 0$

Genes2

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$ G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

relative

row minrow max

Figure S8

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16TFs2iENDO2Characteriza6on2at2day2202

relative row min row max

Annotation ALB AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ hE SC $E nd od er m $D ay $4 $

SOX7$

GATA6$

eGATA4$

GSC$

eMIXL1$

EOMES$

FGF8$

eSOX17$

relative

r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n SOX7

GATA6 eGATA4 GSC

eMIXL1 EOMES FGF8 eSOX17

Genes2

relative r o w m i n r o w m a x

Untransduced hMAPC-Day 20 14TFs derived iENDO-Day 20 hESC- Endodermal progenitors-day 4

A n n o t a t i o n eFOXA2

eFOXA1 CER1 CXCR4 CKIT eHEX

ECADHERIN EPCAM

OCLN eFOXA2$

eFOXA1$

CER1$

CXCR4$

CKIT$

eHEX$

ECADHERIN$

EPCAM$

OCLN$

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $$D ay $2 0$ $ hE SC $E nd od er m $D ay $$4 $

Genes2

ALB$ $ AFP$ $ eHNF4A$

$ eHNF6$

$ PDX1$

relative row min row max

Untransduced hMAPC-Day 20 16TFs derived iENDO-Day 20 hESC diff mature endoderm cells -day 20

Annotation

ALB AFP

eHNF4A eHNF6 PDX1

U TC $MA PC $D ay $2 0$ $ 16 TF s$ iE N D O $D ay $2 0$ $ $ hE SC $L ate $E nd od er m $D ay $2 0$

Genes2

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$

MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$ G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

A)

100μm%

B)

iENDO&HLC&2D&Protocol&B2

Genes%

relative row min row max

iENDO-Day 0 iENDO-HLC.2D hESC-HLC.2D PHH

Annotation ALB

AFP AAT MRP2 CYP3A4 TTR G6PC eHNF4A eHNF6 PROX1 tOCT4 iEND O +D ay% 0% 2D %iEND O +HLC % 2D %hESC +HLC % PHH%

ALB$ AFP$

AAT$ MRP2$

CYP3A4$

TTR$

G6PC$

eHNF4A$

eHNF6$

PROX1$

tOCT4$

Figure S8

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Len$viral*vectors*transduc$on* Endoderm*induc$on*

*****d0**************d1************d3*********d4*********d5*******************************d15********************d20*********

Time%line%for%induced%endodermal%progenitor%(iENDO)%genera8on%from%hMAPC%

MET*clusters*

*forma$on*

hMAPC*medium* Endoderm*induc$on*medium*

Pla$ng**

hMAPC* Viral*vectors**

transduc$on* Repla$ng*

on*matrigel* qRTEPCR/Staining*

Expansion*

Hygromycin*selec$on*

Development of MAPC derived induced endodermal progenitors Sambathkumar, Rangarajan

University of Groningen