Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture.
Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.
Orlandi
Alessia
A
Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Monzino IRCCS, Via Parea 4, 20138 Milan, Italy.
Pagani
Francesca
F
Avitabile
Daniele
D
Bonanno
Giuseppina
G
Scambia
Giovanni
G
Vigna
Elisa
E
Grassi
Francesca
F
Eusebi
Fabrizio
F
Fucile
Sergio
S
Pesce
Maurizio
M
Capogrossi
Maurizio C
MC
D000818:Animals; D000831:Animals, Newborn; D018952:Antigens, CD34 / Q000032:analysis*; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D048430:Cell Shape; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008564:Membrane Potentials; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013234:Stem Cells / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D018952:Antigens, CD34; D050991:GATA4 Transcription Factor; C484170:GATA4 protein, human; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium