Generation of expandable cardiovascular progenitor cells from mouse and human fibroblasts via direct chemical reprogramming

Summary

Here, we present a protocol to reprogram mouse and human fibroblasts into expandable cardiovascular progenitor cells (CPCs) via a defined small-molecule treatment. We describe steps to prepare fibroblasts and generate the chemically induced CPCs (ciCPCs), followed by expansion and differentiation of the ciCPCs. These cells can self-renew in the long term, faithfully retaining the CPC phenotype and cardiovascular differentiation capacity. This protocol provides an autologous and expandable cardiovascular cell source, which may find uses in cardiovascular disease modelling, drug discovery, and cardiac cell therapy.

For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).¹

Graphical abstract

Highlights

• •Direct conversion of fibroblasts into CPCs via a defined small-molecule treatment
• •Detailed procedures for the expansion and differentiation of ciCPCs
• •ciCPCs can be long-term cultured without losing the differentiation capacity
• •Protocol can be used with human and mouse fibroblasts

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to reprogram mouse and human fibroblasts into expandable cardiovascular progenitor cells (CPCs) via a defined small-molecule treatment. We describe steps to prepare fibroblasts and generate the chemically induced CPCs (ciCPCs), followed by expansion and differentiation of the ciCPCs. These cells can self-renew in the long term, faithfully retaining the CPC phenotype and cardiovascular differentiation capacity. This protocol provides an autologous and expandable cardiovascular cell source, which may find uses in cardiovascular disease modelling, drug discovery, and cardiac cell therapy.

Before you begin

Institutional permissions

Human embryonic stem cells and fibroblasts are commercially obtained and all relevant experiments were approved by the Ethical Committee of Sun Yat-sen University and University of Health and Rehabilitation Sciences and comply with the 2021 Guidelines for Stem Cell Research and Clinical Translation (issued by the International Society for Stem Cell Research, ISSCR). All mouse work in this study was done in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised) and carried out under the supervision of the Sun Yat-sen University Animal Care and Use Committee.

Preparations

Timing: 2–3 h

All cells were cultured in a humidified 37°C incubator supplemented with 5% CO₂. The following medium should be prepared before the initiation of each step of the protocol and pre-warmed at 37°C for 20 min before use. Please refer to key resources table for a comprehensive list of reagents and resources. Please refer to materials and equipment for tables of medium recipes.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-GATA4 (Clone: G-4) Dilution 1:400	Santa Cruz Biotechnology	Cat#SC25310; RRID: AB_627667
Mouse monoclonal anti-ISL1 (Clone: 39.4D5) Dilution 1:50	Developmental Studies Hybridoma Bank	Cat#39.4D5; RRID: AB_2314683
Rabbit monoclonal anti-MEF2C (Clone: D80C1) Dilution 1:200	Cell Signalling Technology	Cat# 5030; RRID: AB_10548759
Purified Mouse anti-Ki67 (Clone: B56) Dilution 1:400	BD Biosciences	Cat# 550609; RRID: AB_393778
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 Dilution 1:1000	Life Technologies	Cat# A11008; RRID: AB_143165
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 Dilution 1:1000	Life Technologies	Cat# A21131; RRID: AB_2535771
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 Dilution 1:1000	Life Technologies	Cat# A21241; RRID: AB_2535810
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 Dilution 1:1000	Life Technologies	Cat# A21141; RRID: AB_2535778
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 Dilution 1:1000	Life Technologies	Cat# A21145; RRID: AB_2535781
Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 Dilution 1:1000	Life Technologies	Cat# A21121; RRID: AB_2535764
Chemicals, peptides, and recombinant proteins
CHIR99021	Selleck	S2924; CAS:1797989-42-4
A83-01	Stemgent	04-0014; CAS: 909910-43-6
GSK126	Selleck	S7061; CAS: 1346574-57-9
Forskolin	Sigma	F6886; CAS: 66575-29-9
CTPB	Sigma	EPS001; CAS: 586976-24-1
AM580	Selleck	S2933; CAS:102121-60-8
INDY	MedChemExpress	HY-108476; CAS: 1169755-45-6
SU5402	Selleck	S7667; CAS:215543-92-3
L-ascorbic acid 2-phosphate magnesium	Sigma	A8960; CAS: 1713265-25-8
IWP2	Selleck	S7085; CAS: 686770-61-6
Monothioglycerol (MTG)	Sigma	M6145; CAS: 96-27-5
Heparin sodium salt	Sigma	H3149; CAS:9041-08-1
Insulin solution human	Sigma	I9278; CAS:11061-68-0
Recombinant Human FGF2	PeproTech	100-18B
Recombinant Human BMP4	R&D Systems	314-BP
Recombinant Human Activin A	R&D Systems	338-AC
Recombinant Human TGF-β1	R&D Systems	240-B
Recombinant Human PDGF-BB	R&D Systems	220-BB
B27 supplement without vitamin A	Gibco	12587-010
Knockout serum replacement	Gibco	10828-010
DMEM (4.5 g/l glucose)	Gibco	11965-092
Knockout DMEM (4.5 g/l glucose)	Gibco	10829-018
DMEM/F12	Gibco	11320-033
TeSR™-E8™ Kit for hESC/hiPSC Maintenance	Stem Cell Technologies	05990
IMDM (4.5 g/l glucose)	Gibco	12440-053
F12 medium	Gibco	11765-054
EGM-2 BulletKit	Lonza	CC-3162
Fetal bovine serum (FBS)	Gibco	10099-141
Phosphate-buffered saline (PBS)	Gibco	10010-049
GlutaMAX	Gibco	35050-061
Nonessential amino acids (NEAA)	Gibco	11140-050
β-mercaptoethanol	Sigma	M6250
Insulin-transferrin-sodium selenite (ITS)	Gibco	41400-045
Chemically defined lipid concentrates	Gibco	11905-031
Recombinant human albumin	Sigma	NIST2925
Xeno-free knockout serum replacement	Gibco	12618-012
Collagenase B	Roche	11088815001
Accutase	Stem Cell Technologies	07920
Vitronectin	Gibco	CTS279S3
Matrigel	BD Biosciences	354277
Gelatin	Sigma	V900863
Experimental models: Cell lines
Mouse embryonic fibroblasts: C57BL/6J	Primary cells	N/A
Mouse tail-tip fibroblasts: C57BL/6J	Primary cells	N/A
Human foreskin fibroblasts line CRL2097	American Type Culture Collection	CRL2097
hESC line H1	WiCell	N/A
Experimental models: Organisms/strains
Mouse: C57BL/6J (Male: 8 weeks)	Sun Yat-Sen University Laboratory Animal Center	N/A
Mouse: C57BL/6J (Female: 10 weeks)	Sun Yat-Sen University Laboratory Animal Center	N/A
Other
0.22-μm Millex-GP filter unit	Millipore	SLGP033RB

Materials and equipment

All medium is prepared using sterile techniques. We recommend that cell culture is performed in a sterile environment without the use of antibiotics.

Fibroblast medium

Reagent	Final concentration	Amount
DMEM (4.5 g/l glucose)	N/A	44 mL
FBS	10%	5 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
Total	N/A	50 mL

Note: Fibroblast medium should be stored at 4°C for up to 1 month and pre-warmed at 37°C for at least 20 min prior to use.

Mouse ciCPC reprogramming medium

Reagent	Final concentration	Amount
Knockout DMEM (4.5 g/l glucose)	N/A	39 mL
FBS	10%	5 mL
Knockout serum replacement	10%	5 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
FGF2	100 ng/mL	5 μg
β-mercaptoethanol	0.1 mM	0.4 mg
L-ascorbic acid 2-phosphate magnesium	50 μg/mL	2.5 mg
CHIR99021	10 μM	0.25 mg
A83-01	1 μM	21 μg
GSK126	1 μM	26 μg
Forskolin	50 μM	1 mg
CTPB	5 μM	0.14 mg
AM580	0.03 μM	527 ng
Total	N/A	50 mL

Human ciCPC reprogramming medium

Reagent	Final concentration	Amount
Knockout DMEM	N/A	43 mL
Xeno-free knockout serum replacement	10%	5 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
ITS	1%	0.5 mL
Chemically defined lipid concentrates	1%	0.5 mL
FGF2	10 ng/mL	0.5 μg
β-mercaptoethanol	0.1 mM	0.4 mg
Recombinant human albumin	500 μg/mL	25 mg
L-ascorbic acid 2-phosphate magnesium	50 μg/mL	2.5 mg
CHIR99021	10 μM	0.25 mg
A83-01	1 μM	21 μg
GSK126	1 μM	26 μg
Forskolin	50 μM	1 mg
CTPB	5 μM	0.14 mg
AM580	0.03 μM	527 ng
Total	N/A	50 mL

Note: Store FGF2 stock solution at −80°C for up to 6 months. Then prepare enough 0.1 mL diluted aliquots and store them at −20°C until use. Avoid repeated freezing and thawing. Prepare fresh mouse and human reprogramming medium and filter-sterilize through a 0.2 μm filter before use.

Mouse ciCPC induction medium

Reagent	Final concentration	Amount
DMEM/F12	N/A	48.5 mL
B27 supplement without vitamin A	1%	0.5 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
β-mercaptoethanol	0.1 mM	0.4 mg
CHIR99021	3 μM	75 μg
SU5402	2 μM	29.6 μg
BMP4	5 ng/mL	0.25 μg
Activin A	10 ng/mL	0.5 μg
Total	N/A	50 mL

Human ciCPC induction medium

Reagent	Final concentration	Amount
DMEM/F12	N/A	48 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
ITS	1%	0.5 mL
Chemically defined lipid concentrates	1%	0.5 mL
β-mercaptoethanol	0.1 mM	0.4 mg
Recombinant human albumin	500 μg/mL	25 mg
CHIR99021	3 μM	75 μg
SU5402	2 μM	29.6 μg
BMP4	5 ng/mL	0.25 μg
Activin A	10 ng/mL	0.5 μg
Total	N/A	50 mL

Note: Store BMP4 and Activin A stock solution at −80°C for up to 6 months. Then prepare enough 0.1 mL diluted aliquots and store them at −20°C until use. Avoid repeated freezing and thawing. Prepare fresh mouse and human induction medium and filter-sterilize through a 0.2 μm filter before use.

Mouse and human ciCPC expansion medium

Reagent	Final concentration	Amount
DMEM/ F12	N/A	48 mL
NEAA	1%	0.5 mL
GlutaMAX	1%	0.5 mL
ITS	1%	0.5 mL
Chemically defined lipid concentrates	1%	0.5 mL
L-ascorbic acid 2-phosphate magnesium	64 μg/mL	3.2 mg
CHIR99021	3 μM	75 μg
SU5402	2 μM	29.6 μg
BMP4	5 ng/mL	0.25 μg
Activin A	10 ng/mL	0.5 μg
INDY (human ciCPC expansion medium only)	0.2 μM	2.35 μg
Total	N/A	50 mL

Note: Make fresh ciCPC expansion medium and filter-sterilize through a 0.2 μm filter. For long-term expansion of human ciCPCs, INDY should be added to promote cell growth.

Mouse cardiomyocyte (CM) differentiation medium

Reagent	Final concentration	Amount
IMDM (4.5 g/l glucose): F12	3:1	49 mL
B27 supplement without vitamin A	1%	0.5 mL
GlutaMAX	1%	0.5 mL
L-ascorbic acid 2-phosphate magnesium	50 μg/mL	2.5 mg
MTG	0.4 mM	2.2 mg
IWP2	5 μM	0.12 mg
Total	N/A	50 mL

Note: Mouse CM differentiation medium should be stored at 4°C for up to 1 week and pre-warmed at 37°C for at least 20 min prior to use.

Mouse smooth muscle cell (SMC) differentiation medium

Reagent	Final concentration	Amount
IMDM (4.5 g/l glucose): F12	3:1	49 mL
B27 supplement without vitamin A	1%	0.5 mL
GlutaMAX	1%	0.5 mL
L-ascorbic acid 2-phosphate magnesium	50 μg/mL	2.5 mg
MTG	0.4 mM	2.2 mg
PDGF-BB	10 ng/mL	0.5 μg
TGF-β1	2 ng/mL	0.1 μg
Total	N/A	50 mL

Note: Store PDGF-BB and TGF-β1 stock solution at −80°C for up to 6 months. Then prepare enough 0.1 mL diluted aliquots and store them at −20°C until use. Avoid repeated freezing and thawing. SMC differentiation medium should be filter-sterilized through a 0.2 μm filter before use and stored at 4°C for up to 1 week.

Human ciCPC CM differentiation medium

• •Conditioned medium from human embryonic stem cell (hESC)-derived CMs supplemented with 5 μM IWP2.

Note: Conditioned medium should be stored at −80°C until use and should be used within 2 weeks after thawing.

CRITICAL: Detailed procedures of conditioned medium collection will be described in the “step-by-step method details: 27. Cardiac differentiation of hESCs and conditioned medium preparation” section as listed below.

hESC CM differentiation medium

Reagent	Final concentration	Amount
E8 basal medium	N/A	49.5 mL
Chemically defined lipid concentrates	1%	0.5 mL
CHIR99021 (added during day 0-day 1)	5 μM	0.13 mg
IWP2 (added during day 2-day 5)	3 μM	70 μg
Heparin (added during day 2-day 5)	3 μg/mL	0.15 mg
Insulin (day 7 onwards)	20 μg/mL	1 mg
Total	N/A	50 mL

Note: hESC CM differentiation medium should be stored at 4°C for up to 1 week.

Human CM culture medium

Reagent	Final concentration	Amount
DMEM/ F12	N/A	48 mL
ITS	1%	0.5 mL
Chemically defined lipid concentrates	1%	0.5 mL
L-ascorbic acid 2-phosphate magnesium	64 μg/mL	3.2 mg
Total	N/A	50 mL

Note: Human CM culture medium should be stored at 4°C for up to 2 weeks.

Step-by-step method details

Fibroblast preparation

Timing: 4 days

This step describes methods to prepare and culture mouse and human fibroblasts.

• 1.
  Prepare mouse and human fibroblast cells.

  • a.
    To obtain healthy and proliferative mouse fibroblast cells for reprogramming, mouse embryonic fibroblasts (MEFs) or tail-tip fibroblasts (TTFs) are derived from 13.5-day mouse embryos or 8-weeks adult mice, respectively, according to a standard procedure.² MEFs and TTFs are maintained in fibroblast medium and cells that have been passaged for less than 3 times are used.
  • b.
    Human foreskin fibroblast (HFF) line CRL2097 are used for reprogramming and are passaged onto the gelatin-coated dishes in fibroblast medium (troubleshooting 1).

CRITICAL: For MEF preparation, remove head, spinal cords, and all developing organs from mouse embryos to avoid lineage precursor cell contamination.

ciCPC induction

Timing: 15–27 days

This step describes the stepwise procedure to directly generate ciCPCs from mouse or human fibroblasts.

• 2.Coat a 12-well plate with matrigel (for mouse ciCPC induction) or a 6-well plate with vitronectin (for human ciCPC induction) and place the plate in a 37°C, 5% CO₂ tissue culture incubator for at least 1 h (troubleshooting 1).
• 3.Carefully remove the coating reagent and plate 10,000 (MEFs), 20,000 (TTFs), or 40,000 (HFFs) cells per well, respectively, in pre-warmed fibroblast medium.
• 4.Place the plate in a 37°C, 5% CO₂ tissue culture incubator for at least 8 h.

CRITICAL: Fibroblasts are likely to aggregate. As evenly distributed cells and appropriate cell confluence are both important for reprogramming, therefore rock the plate gently to evenly distribute fibroblast cells.

• 5.Carefully aspirate the fibroblast medium without touching the bottom of the plate and wash with PBS twice.
• 6.Add 2 mL of fresh mouse ciCPC reprogramming medium or 2.5 mL of human ciCPC reprogramming per well, respectively, which includes a combination of 6 compounds (6C) comprising CHIR99021, A83-01, GSK126, Forskolin, CTPB, and AM580 for CPC induction.

CRITICAL: It is important not to disturb the attached cells which may result in low reprogramming efficiency. Therefore, gently and slowly add medium along the side wall of the well and avoid adding medium directly onto the cultured cells.

• 7.Place the plate in a 37°C, 5% CO₂ tissue culture incubator. Replace with fresh reprogramming medium every 2 days.
• 8.After 8 (for mouse ciCPC induction) or 20 days (for human ciCPC induction) of incubation, carefully aspirate the reprogramming medium from the plates and wash with PBS twice (troubleshooting 3).
• 9.Add 2 mL of fresh mouse or human ciCPC induction medium per well, respectively, and return the plates to the incubator.
• 10.Change medium every 2 days.
• 11.After 6 days, mouse (Figure 1) or human (Figure 2) ciCPC colonies are formed and can be picked for further expansion (troubleshooting 4).

CRITICAL: Compared to mouse ciCPC reprogramming condition, HFFs are executed to chemical-treatment for continuous 20 days due to slower growth rate and reprogramming kinetics of human fibroblasts. 6C-treating time may vary from 15–30 days for different fibroblast lines and optimal treating duration for each line may require experimental validation.

Figure 1.

Reprogramming of MEFs into CPCs via defined small-molecule treatment

(A) Morphology of 6C-induced cell colonies derived from MEFs at day 14. Scale bars, 100 μm.

(B) Immunofluorescence analyses of Gata4 and Isl1 in 6C-induced cell colonies at day 14. Scale bars, 100 μm.

Figure 2.

Reprogramming of HFFs into CPCs via defined small-molecule treatment

(A) Morphology of 6C-induced cell colonies derived from HFFs at day 26. Scale bars, 100 μm.

(B) Immunofluorescence analyses of GATA4 and ISL1 in 6C-induced cell colonies at day 26. Scale bars, 100 μm.

ciCPC clone picking and expansion

Timing: 2–3 h

This step describes the methods for picking the ciCPC clones and subsequent long-term expansion of them in a chemically defined xeno-free condition.

• 12.Pick mouse or human ciCPC colonies into a 1.5 mL-tube.
• 13.Centrifuge the picked colonies at 800 g for 3 min at 25°C.
• 14.Carefully remove the supernatant, resuspend the pellet in 0.1 mL collagenase B, and digest for 10 min at 37°C.
• 15.Quench the enzyme by adding 0.1 mL of fresh ciCPC expansion medium and centrifuge the digested cells at 800 g for 3 min at 25°C.
• 16.Carefully remove the supernatant and resuspend the picked colonies in 0.1 mL of accutase and digest for 5 min at 37°C.
• 17.Repeat procedure 15. Then carefully remove the supernatant and resuspend the digested cells in 0.1 mL of fresh ciCPC expansion medium and seed them into a well of a vitronectin-coated 96-well plate.

CRITICAL: Do not rock the plate as cells in 96-well plate tend to gather in the well center upon shaking.

• 18.Cultured ciCPCs are ready for passaging when grow to confluence. For passaging, aspirate the ciCPC expansion medium, and wash the cells twice with PBS. Add 0.1 mL collagenase B per well of 96-well plate and digest for 10 min at 37°C (troubleshooting 5).
• 19.Carefully remove collagenase B without dislodge the cells, and add 0.1 mL accutase per well of 96-well plate for 5 min at 37°C.
• 20.Quench the enzyme by adding 0.1 mL of fresh ciCPC expansion medium into each well.
• 21.Collect all undetached cells from the well and transfer them into a 15-mL tube. Then centrifuge the cells at 800 g for 3 min at 25°C.
• 22.Carefully remove the supernatant and resuspend the cells in 1 mL of fresh expansion medium. Pipette the cells up and down thoroughly.
• 23.Add the single-cell suspensions into the vitronectin-coated 24-well plate. Gently rock the plate to evenly distribute the ciCPCs and then place the plate in a 37°C, 5% CO₂ tissue culture incubator (troubleshooting 1).
• 24.For long-term expansion, cells can be routinely passaged every 3–4 days (for mouse ciCPCs) or 4–6 day (for human ciCPCs), respectively, at a density of 5×10⁵ cells per well of a 12 well plate. The culture medium should be changed every 2 days (troubleshooting 5).
• 25.In this defined culture condition, mouse or human ciCPCs can long-term propagate for more than 20 or 12 passages, respectively, with stable growth rate and undifferentiated morphology maintained (Figure 3).

Pause point: Mouse and human ciCPCs can be frozen and long-term stored in liquid nitrogen.

Figure 3.

ciCPCs are long-term expandable in fully chemically defined conditions

(A) Morphology of P20 mouse ciCPCs. P, passage. Scale bars, 100 μm.

(B) Morphology of P12 human ciCPCs. Scale bars, 100 μm.

(C) Immunofluorescence analyses of Ki67, Gata4, and Isl1 in P20 mouse ciCPCs. Scale bars, 100 μm.

(D) Immunofluorescence analyses of MEF2C and GATA4 in P12 human ciCPCs. Scale bars, 100 μm.

ciCPC differentiation

Timing: 7–14 days

This step describes the methods for targeted differentiation of ciCPCs into cardiomyocytes (CMs), smooth muscle cells (SMCs), endothelial cells (ECs).

• 26.
  Procedures of mouse ciCPC differentiation in vitro (troubleshooting 1 and 6).

  • a.
    For CM differentiation, plate accutase-dissociated mouse ciCPCs onto the vitronectin-coated plate in fresh mouse CM differentiation medium for continuous 7 days (at a density of 3×10⁵ cells per cm²). Change medium every 2 days.
  • b.
    CMs display synchronized contraction usually at days 7.
  • c.
    For SMC differentiation, suspend accutase-dissociated mouse ciCPCs with fresh SMC differentiation medium.
  • d.
    Add the suspension to the vitronectin-coated plate at a density of 1×10⁴ cells per cm².
  • e.
    Culture for 7 days. Change medium every 2 days. Differentiated SMCs can be monitored at day 7.
  • f.
    For EC differentiation, plate accutase-dissociated mouse ciCPCs onto the vitronectin-coated plate in EGM-2 medium for continuous 7 days (at a density of 1×10⁴ cells per cm²). Change medium every 2 days.
  • g.
    Differentiated ECs can be monitored at day 7.

Pause point: Cells can be fixed and stored in PBS at 4°C for 2–3 weeks.

• 27.
  Cardiac differentiation of hESCs and conditioned medium preparation.

  • a.
    hESC line H1 is cultured on the vitronectin-coated plate in TeSR™-E8™ medium (troubleshooting 1).
  • b.
    Cardiac differentiation of H1 hESCs is initiated by seeding hESCs in monolayer in TeSR™-E8™ medium and allowing them to grow to 80%–90% confluence.
  • c.
    Switch to hESC CM differentiation medium and name this day as day 0. The medium should be changed daily until day 7 and every 2–3 days thereafter.
  • d.
    Needed supplements in CM differentiation medium are varied in different differentiation days. Add correct supplement at the indicated dosages and times following the recipe listed above.
  • e.
    On differentiation day 20, reseed cells onto the gelatin-coated plate in human CM culture medium.
  • f.
    Collect the conditioned medium of the differentiated CMs from differentiation day 30 to day 60. Medium is harvested every 2 days.

CRITICAL: Split the conditioned medium into 15-mL tubes. It should be stored at −80°C until use and should be used within 2 weeks after thawing.

• 28.
  Procedures of human ciCPC differentiation in vitro (troubleshooting 1 and 6).

  • a.
    For CM differentiation, plate accutase-dissociated human ciCPCs onto the vitronectin-coated plate in fresh human ciCPC CM differentiation medium for 14 days (at a density of 3×10⁵ cells per cm²). Culture medium is renewed every 2 days.
  • b.
    Procedures of the SMC and EC differentiation from human ciCPCs are the same as the mouse ciCPCs except extending the treating time to 14 days.

CRITICAL: Quality of conditioned medium of hESC-derived CMs is very important for cardiac differentiation of human ciCPCs due to its paracrine factors. Use freshly thawed conditioned medium for CM differentiation of human ciCPCs.

Pause point: Cells can be fixed and stored in PBS at 4°C for 2–3 weeks.

Expected outcomes

CPCs are lineage-restricted progenitors that have extensive proliferative capacity and multi-potency towards cardiovascular lineages (including CMs, SMCs and ECs).³ Compared to CMs, CPCs display a greater potency for heart repairing after transplantation into the mouse myocardial infarction heart.^4,5,6,7 In this protocol, we describe an efficient method for generation of expandable CPCs from fibroblasts with defined small molecules.

By stimulation with the reprogramming small molecule cocktail, mouse and human fibroblasts are initially induced to an epigenetically unstable cell state with increased plasticity. Then the activated cells are specifically directed towards a CPC state with the treatment of defined small molecules and cytokines. Following this protocol, fibroblasts undergo morphology changes and convert into Gata4⁺/Isl1⁺ ciCPCs (Figures 1 and 2). In terms of long-term expansion, ciCPCs can robustly propagate for more than 20 passages (mouse) and 12 passages (human), respectively, in the optimized culture condition. Further characterization assays reveal that late passage ciCPCs retain undifferentiated morphology, stable growth rate, and uniform expression of CPCs markers (Figure 3). Passaged ciCPCs can efficiently differentiate into cardiovascular lineages cells.

In summary, this protocol enables ciCPC self-renewal long-term in vitro. Expanded ciCPCs can be frozen stored and thawed for rapid use.

Limitations

This protocol provides a method for reprogramming of fibroblasts into expandable CPCs without introducing genetic manipulation.¹ Nevertheless, there are still limitations that should be overcome before the method can be widely used for future clinical translation.

First, human CPC reprogramming is relatively inefficient (∼5% based on key marker expression) and time consuming, which may result from the slower growth rate and reprogramming kinetics of HFFs. The current protocol should be optimized through additional boosting molecule screening to improve human reprogramming efficiency.

Secondly, in this protocol human fetal fibroblasts are used as the starting population for reprogramming, as they are more epigenetically amenable. Generating ciCPCs from adult human fibroblasts are still challenging. Identification of additional small molecules that enables adult human somatic cells is a prerequisite for further optimization of the current reprogramming protocol and future clinical application.

Troubleshooting

Problem 1

Poor attachment of cells in the coated plate (related to steps 1, 2, 23, 26, 27, and 28).

Potential solution

It may be caused by imperfect substrate coating. We recommend restarting with a new properly coated plate. For matrigel coating, it is important to work on ice and use cold tips. Besides, perform medium changing carefully and gently.

Problem 2

Cultures become contaminated (related to each step).

Potential solution

To avoid contamination, operation should be always performed in the sterile culture hood. All the medium should be filter-sterilized through 0.2 μm filter before use.

Problem 3

Mouse ciCPC colonies fail to appear during the reprogramming process (related to step 8).

Potential solution

It may be caused by poor quality of the starting fibroblasts which may affect the reprogramming efficiency and final outcomes. To ensure a healthy and proliferative starting population, we don’t recommend using frozen cells or fibroblasts passaged greater than 3 times for mouse ciCPCs reprogramming. In addition, fine-tuning of the concentration of each reprogramming chemical may be required for some type of mouse fibroblasts.

Problem 4

Human ciCPC colonies are not observed after induction (related to step 11).

Potential solution

There are two common reasons for this problem: 1) Freshly thawed HFFs are used immediately for reprogramming; 2) Small molecules or cytokines used in the medium are improperly stored.

To initial with an active starting population, we recommend use HFFs passaged at least one time after thawing, and HFFs passaged greater than 10 should not be used for ciCPC reprogramming. Secondly, small molecules or cytokines aliquots should be stored at the correct temperature and used within the indicated time. Avoid repeated freeze-thaw cycles and use freshly prepared medium.

Problem 5

Low survival rate during ciCPC seeding and expansion (related to steps 18 and 24).

Potential solution

Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors, such as thiazovivin (1 μM, Stemgent) or Y27632 (10 μM, Stemgent), can be added to the expansion medium to improve cell survival.

Problem 6

Low differentiation efficiency of mouse and human ciCPCs in vitro (related to steps 26 and 28).

Potential solution

The most common reason for this problem is the unsuitable cell density during initial cell seeding which may negatively affect the differentiation efficiency. To harvest specifically differentiated cells maximally, we strongly recommend fine-tuning the initial cell seeding number around the standard density.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nan Cao (caon3@mail.sysu.edu.cn).

Materials availability

This study did not generate any unique reagents.

Data and code availability

This study did not generate/analyze any datasets or code.