Isolation and Expansion of Adult Cardiac Stem/Progenitor Cells in the Form of Cardiospheres from Human Cardiac Biopsies and Murine Hearts

Abstract

The successful isolation and ex vivo expansion of resident cardiac stem/progenitor cells from human heart biopsies has allowed us to study their biological characteristics and their applications in therapeutic approaches for the repair of ischemic/infarcted heart, the preparation of tissue-engineered cardiac grafts and, possibly, the design of cellular kits for drug screening applications. From the first publication of the original method in 2004, several adjustments and slight changes have been introduced to optimize and adjust the procedure to the evolving experimental and translational needs. Moreover, due to the wide applicability of such a method (which is based on the exploitation of intrinsic functional properties of cells with regenerative properties that are present in most tissues), the key steps of this procedure have been used to derive several kinds of tissue-specific adult stem cells for preclinical or clinical purposes.In order to define the original procedure, complete with the up-to-date modifications introduced through the years, an exhaustive description of the current protocol is performed in this chapter, with particular attention in highlighting critical steps and troubleshoots. The procedure described here consists of modular steps, that could be employed to derive cells from any kind of tissue biopsy, and needs to be considered the gold standard of all the so-called "explant methods" or "cardiosphere methods," and it represents a milestone in the clinical translation of autologous cell therapy.

Spheroid three-dimensional culture enhances Notch signaling in cardiac progenitor cells

“…Adult explant-derived cells (EDCs) were isolated from human surgical auricular biopsies, as previously described. [6,7] Once 80% of confluency was reached, EDCs were collected and seeded on poly-D-lysine-coated 12-multiwell plates with a cell density of 9 × 10 3 cells/cm 2 to form CSps. CMPC-CSps and CSps spontaneously formed after 5 days of culture in either room air (20% O 2 ) or physiologic (5% O 2 ) oxygen conditions, and collected at this time-point.…”

“…Cardiospheres (CSps) are cardiac-derived multicellular spheroids, spontaneously formed by cells migrating from cardiac tissue explants and subsequently cultured on a poly-D-lysine-coated dish. [6,7] CSps can serve as an interesting in vitro model of the cardiac stem cell niche. [8] Compared with traditional two-dimensional (2D) culture, multicellular spheroids provide a microenvironment that more closely mimics the in vivo physiology.…”

An epigenetic perspective on the failing heart and pluripotent-derived-cardiomyocytes for cell replacement therapy

“…For the latter, there is evidence to support endogenous cardiomyogenesis in mice and humans (Bergmann et al, 2009;Senyo et al, 2013) including MI induction of CMs to re-commence cell division in mice (Porrello et al, 2011;Senyo et al, 2013). Further, recent work has established the presence of, and isolation protocols for, CPs existing in specific niches, capable of proliferation and CM differentiation (Leri et al, 2011;Chimenti et al, 2012). Despite these encouraging results, endogenous CM sources for cell replacement currently remain hugely inadequate in the face of large-scale cellular losses that accompany acute MI and progressive remodeling during HF (Zwi-Dantsis and Gepstein, 2012).…”

TGFβ-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies

“…Human auricolar biopsies (10 patients) were cultivated as explants, and CPCs were isolated with the CSp protocol, as previously described [5,22]. Briefly, EDCs were collected weekly up to 3 times from each explant, and seeded on poly-D-lysine (BD-Biosciences) coated wells (9,000 cells/cm 2 ) to obtain CSps.…”