In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Summary

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Abstract

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.

Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so.

Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

Introduction

Congestive heart failure (CHF) persists as a leading cause of morbidity and mortality worldwide. CHF often occurs following the massive loss of cardiomyocytes and the development of cell-free scar tissue as the pathological result of a myocardial infarction (MI)¹. While the heart is a partially self-renewing organ, the resident stem and progenitor cell pool responsible for executing tissue regeneration significantly diminishes in abundance and function in aged patients, often becoming insufficient for optimal recovery after injury. Thus, there is great interest in developing experimental treatments that involve the transplantation of healthy do....

Protocol

All studies involving animals were conducted and reported according to ARRIVE guidelines⁴⁸. All studies were performed with institutional research ethics board approval (REB number 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8^th edition (National Institutes of Health 2011).

1. Tissue Culture

1. Culture FTM HUCPVCs, term HUCPVC....

Results

HUCPVCs Display Higher Aggregate-formation Potential and CD49f Expression Levels Compared to BMSCs:

To induce the differentiation of hMSCs (i.e., FTM HUCPVCs, term HUCPVCs, and BMSCs), single-cell suspensions of undifferentiated MSCs or MSC-containing hanging drops (Table 1) were transferred onto rat primary cardiomyocyte monolayers to establish direct co-cultures or aggregate co-cultur.......

Discussion

The cardiac differentiation of stem cells has been under development for over 2 decades, with several different strategies being used to generate cardiomyocyte-like cells from MSC sources. Many of these strategies, however, are inefficient, and the conditions used are often not representative of the environment transplanted cells encounter in vivo.

In contrast to existing methods, the protocol presented here utilizes a combination of primary cardiac feeder layers and MSC aggregate for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin/EDTA	Gibco	25200056	For cell dissociation
Alpha-MEM	Gibco	12571071	For HUCPVC and BMSC culture media.
PE-conjugated anti-human/mouse CD49f antibody	Biolegend	313612	Integrin marker for FC
APC-conjugated human Cx43/GJA1 antibody	R&D Systems	FAB7737A	Connexin 43 marker for FC
FITC-conjugated HLA-A2 antibody	Genway Biotech Inc.	GWB-66FBD2	Immunogenicity marker for FC
FITC-conjugated anti-HLA-G [MEM-G/9] antibody	Abcam	ab7904	Immunogenicity marker for FC
FITC-conjugated mouse anti-human SIRPA/CD172a antibody	AbD Serotec/Bio-Rad	MCA2518F	Cardiac marker for FC
APC-conjugated human TRA-1-85/CD147 antibody	R&D Systems	FAB3195A	Human cell marker for FC and FACS
FITC-conjugated human TRA-1-85/CD147 antibody	R&D Systems	FAB3195F	Human cell marker for FC and FACS
Anti-connexin 43/GJA1 antibody	Abcam	ab11370	Cx43. For ICC
Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555	Life Technologies	A-21428	For ICC
Anti-sarcomeric alpha actinin [EA-53] antibody	Abcam	ab9465	aSARC. For ICC
Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555	Life Technologies	A-21426	For ICC
Mef2C (D80C1) XP rabbit antibody	New England BioLabs Ltd.	5030S	For ICC
Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488	Life Technologies	A-21206	For ICC
Anti-nuclei (HuNu) (clone 235-1) antibody	EMD Millipore	MAB1281	For ICC
MZ9.5 Stereomicroscope	Leica		For imaging aggregates.
1.5 ml centrifuge microtubes	Axygen	MCT-150-C	For staining MSCs with fluorescent dye.
ImageJ			Open source image processing software.
Aria II	BD		UHN SickKids FC Facility. For cell sorting.
Bone marrow mesechymal stromal cells	Lonza	PT-2501	BMSCs
Bovine serum albumin	Sigma-Aldrich	A7030-100G	BSA. To prepare solutions for ICC
BrdU	EMD Millipore	MAB3424	Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.
Canto II	BD		UHN SickKids FC Facility. For flow cytometry.
cDNA EcoDry Premix	Clontech/Takara	639570	For preparation of cDNA for qPCR
CellTracker Green CMFDA Dye	Life Technologies	C7025	Fluorescent imaging of cell cytoplasm
Countess automated cell counter	Invitrogen Inc.	C10227	For cell counting
DMEM-F12	Sigma-Aldrich	D6421	For rat primary cardiomyocyte culture medium.
Dulbecco's Phosphate Buffered Saline	Gibco	10010023	D-PBS, without Ca2+, Mg2+
EVOS	Life Technologies		In-house fluorescent microscope
FACSCalibur	BD		In-house. For flow cytometry.
Fetal bovine serum (Hyclone)	GE Healthcare	SH3039603	FBS. Component of cell culture medium.
IDT Prime Time qPCR probes	Integrated Data Technologies	FAM fluorophore	http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers
Lab Vision PermaFluor Aqueous Mounting Medium	ThermoScientific	TA-030-FM	For storage of cells to undergo ICC
LSR II	BD		UHN SickKids FC Facility. For flow cytometry.
MoFlo Astrios	Beckman Coulter		UHN SickKids FC Facility. For cell sorting.
Normal goat serum	Cell Signaling Technology	5425S	NGS. Used in blocking solution for ICC
Nunc Lab-Tek II Chamber Coverglass, 8-wells	Thermo Scientific Nunc	155409	To prepare samples for ICC
OmniPur Triton X-100 Surfactant	EMD Millipore	9410-OP	As a component of permeabilizing solution when preparing cells for ICC
Paraformaldehyde, 16% Solution, EM Grade	Electron Microscopy Sciences	15710	For fixing cells for ICC.
Penicillin/streptomycin	Gibco	15140122	Component of cell culture medium.
Primers	Sigma	Custom Standard DNA Oligos, Desalted, 0.2 μmol	CTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G
Quorum Spinning Disk Confocal	Zeiss		SickKids Imaging Facility
ReproCardio hiPS cell derived cardiomyocytes	ReproCell	RCD001N	Positive control for qPCR
RNeasy mini kit	Qiagen	74106	To isolate RNA for qPCR
Rotor-Gene SYBR Green PCR Kit	Qiagen	204074	For qPCR with master mix
RPMI 1640	Gibco	A1049101	For MSC, monocyte coculture medium.
TaqMan qPCR primer assays	Thermo Fisher Scientific	4444556	For qPCR
Trypan Blue	Life Technologies	T10282	Staining of cells for viability and counting
Trypsin	Gibco	272500108	For cell dissociation
Volocity	Perkin-Elmer	Volocity 6.3	Imaging software
0.2 μm pore filter	Thermo Fisher Scientific	566-0020	For sterilizing tissue culture media
HERAcell 150i CO2 Incubator	Thermo Fisher Scientific	51026410	For incubating cells
Dulbecco's phosphate buffered saline	Sigma-Aldrich	D8537	PBS. 1X, Without calcium chloride and magnesium chloride
Forceps	Almedic	7727-A10-704	For handing rat heart. Can use any similar forceps.
Scissors	Fine Science Tools	14059-11	For mincing rat heart. Curved scissors recommended.
50 mL tube	BD Falcon	352070	For collection during cardiomyocyte collection and general tissue culture procedures
15 mL tube	BD Falcon	352096	For general tissue culture procedures
6-well plates	Thermo Scientific Nunc	CA73520-906	For tissue culture
10 cm tissue culture dishes	Corning	25382-428	For aggregate formation
Axiovert 40C Microscope	Zeiss		For bright-field imaging through out tissue culture and the rest of the protocol
70 μm cell strainer	Fisherbrand	22363548	To ensure a single cell suspension before flow cytometry or sorting
Triton X-100	EMD Millipore	9410-1L	Used in permeabilization solution for ICC
Hoechst 33342	Thermo Fisher Scientific	H1399	Stain used during visualization of Cx43 localization