This document describes a method for reprogramming fibroblasts into cardiovascular progenitor cells (ciCPCs) using small molecules. The goals are to develop an autologous source of CPCs, attain high proliferation capacity, and generate a multipotent but cardiovascular-restricted cell type. Mouse and human fibroblasts were reprogrammed into ciCPCs expressing cardiac progenitor markers using a 6-day chemical cocktail. The ciCPCs could be expanded and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo, demonstrating their potential for cardiac repair applications. However, limitations include potential immunogenicity and a low reprogramming efficiency in human cells.

1. Reprogramming
Fibroblasts into
Cardiovascular
Progenitors via Small
Molecules in Xeno-free
Conditions
Aparna Sinha
(26 May 2022)

2. Cardiac Regeneration
Flk1 VEGFR
Bry Regulates posterior mesoderm
and axial development
MESP1 Activates TF for cardiac
development
ISL1 2nd wave CPC marker. Activates
TF for cardiac development
(eg.GATA4)
GATA4 morphogenesis of the
precardiogenic mesodermal
cells for cardiac development
NKx2.5 Regulates cardiac
morphogenesis and actives TFs
for cardiac development
⍺SMA Smooth muscle actin
Calponin Actin filament marker
cTNT Cardiac troponin T- regulates
actin myosin interaction

4. Aims
To develop an autologous source of CPCs
To attain a high proliferation capacity and
expandability of the CPCs
To get a multipotent but cardiovascular lineage
restricted cell type

5. ciCPC induction with 6C

6. ciCPC induction with 6C

7. ciCPC expansion via BACS(I)

8. BACS(I)
• BMP4
• Activin A
• CHIR99021- GSK3 inhibitor
• SU5402- inhibits VEGF, FGF, PDGF
• INDY (dual-specificity tyrosine phosphorylation-regulated kinase
(DYRK) inhibitor)- increases cell proliferation by two folds in hciCPCs

9. Chemically induced CPCs (ciCPCs) in mice
Culture for 6
days and expand

10. Reprogramming of mciCPCs by 6C
Figure: mmunofluorescence analyses of
Gata4 and isl1
in 6C-induced cell colonies at day 14. Scale
bars, 100 μm.

11. Reprogramming of mciCPCs by 6C
Figure: Flow-cytometric analysis
of the percentage of
Flk1+/PdgfRα+ ciCPCs with or
without 6C treatments (n = 3
biologically independent
experiments). Horizontal and
vertical lines indicate the
boundaries of cells that are
negative or positive for the
markers stained.

12. ciCPCs are genetically stable
Immunofluorescence analyses of CPC marker Gata4, isl1, Nkx2-5 and Mef2c
and proliferative marker Ki67, in P3 and P20 ciCPCs. Scale bars, 100 μm.
Flow-cytometric analysis of the percentage of
Flk1+/PdgfRα+ ciCPCs at P3 and P20 (n = 3
biologically independent experiments)

13. ciCPCs have tri-lineage potency
P20

14. ciCPC derived Cardiomyocytes
Figure: (a) Flow-cytometric analysis of the percent of cTnT+ CMs in P20 ciCPCs that underwent a 7- d cardiac differentiation (n
= 3 biologically independent experiments). The inset box represents the cTnT+ CMs. (b) Transmission electron microscopic
analyses of ciCPC-CMs. Myofilaments (blue arrows), Z-bands (red arrows) and mitochondria (yellow arrows) are visible. Scale
bars, 500 nm. (c) Beating cluster generated after MEF-derived ciCPCs were cultured in cardiac differentiation conditions for
7 d.
a b c

15. ciCPC derived Smooth Muscle Cells
Figure: immunofluorescence analyses reveal the expression of SMC markers in ciCPC-SMCs. Scale bars, 100 μm

16. ciCPC derived Endothelial Cells
Figure: immunofluorescence analyses reveal the expression of EC markers in ciCPC-ECs. Scale bars, 100 μm

17. ciCPCs repair heart in vitro after MI

18. ciCPCs repair heart in vitro after MI
Figure: (a) Gross appearance of transplanted ciCPCs revealed by immunofluorescence analyses of nuclear-localizing GFP
(nlGFP, top) and CM marker cTnT (bottom). (b) immunofluorescence analyses of nlGFP and CM (left), SMC (middle) and EC
(right) markers in tissue sections in a. Scale bars, 25 μm.

19. Chemically induced CPCs (ciCPCs) in humans

20. Chemically induced CPCs (ciCPCs) in humans
Figure: immunofluorescence analyses of CPC markers on cell colonies induced from HFFs by 6C at day 26. Scale bars, 100 μm.

21. Chemically induced CPCs (ciCPCs) in humans
Figure: Flow-cytometric analysis of the percentage of
KDR+/PDGFRα+ hciCPCs at P2 and P12 (n = 3
biologically independent experiments).
KDR+/PDGFRα+ cells are enclosed by the black
circles and their percentages are labelled in red.

22. hciCPCs have trilineage potency in vitro

23. hciCPCs have trilineage potency in vitro
Figure: (a) immunofluorescence analyses of CM markers in human ciCPC-CMs (hciCPC-CMs) (n = 3 biologically independent
experiments). Scale bars, 100 μm. (b) Beating cluster generated after HFF-derived ciCPCs were cultured in cardiac
differentiation conditions for 14 d.

24. hciCPCs have trilineage potency in vitro
Figure: Immunofluorescence
analyses of EC markers in human
ciCPC-ECs (n = 3 biologically
independent experiments). Scale
bars, 100 μm
Day 14 of culture in differentiation
medium

25. hciCPCs have trilineage potency in vitro
Figure: Immunofluorescence analyses of SMC (g) markers in
human ciCPC-SMCs (n = 3 biologically independent
experiments). Scale bars, 100 μm
Day 14 of culture in differentiation medium

26. hciCPCs have trilineage potency in vivo

27. hciCPCs have trilineage potency in vivo
Figure: immunofluorescence
analyses of human specific
Lamin A/C and CM (left), SMC
(middle) or EC (right) markers
in tissue sections 2 weeks after
transplantation differentiated
into cTNT+ CMs, calponin+
SMCs or CD31+ ECs

28. Advantages
• Easy to translate for clinical trials
• Differentiate into the three CV cell types to improve heart repair
• Rely on paracrine signalling in vivo for differentiation
• Non-tumorigenic and not genetically modified
• Can be used for drug trials

29. Limitations
• Caused immunogenic response in immune-competent mice
• Cell source for humans not defined
• For humans 5.5% cells attained KDR+PDGFR⍺+ phenotype till day
26 of culture
• Time constraints?