Hirata N., Nakagawa M., Fujibayashi Y., Yamauchi K., Murata A., Minami I., Tomioka M., Kondo T., Kuo T-F., Endo H., Inoue H., Sato S., Ando S., Kawazoe Y., Aiba K., Nagata K., Kawase E., Chang Y-T., Suemori H., Eto K., Nakauchi H., Yamanaka S., Nakatsuji N., Ueda K., Uesugi M.
Cell Reports, 2014
The Yamanaka group uses cardiac specific laminin-211 as the matrix to differentiate iPS cells to cardiomyocytes in a biorelevant environment specific to heart cells. This thus shows that you can use laminin-211 as a cardiac matrix. 326 fluorescent compounds screened to identify a fluorescent probe that is selective for human pluripotent stem cells compared to differentiated cells. hiPSCs were cultured on 3.5 cm culture dishes coated with human laminin 211 and cardiac differentiation was carried out. Cardiac colonies were harvested on day 15 and cultured for 7–10 days in floating culture. A majority of the prepared cells expressed the cardiac markers: cardiac troponin T, a-actinin, and NKX2.5.
Burridge P., Elena Matsa E., Shukla P., Lin Z., Churko J., Ebert A., Lan F., Diecke S., Huber B., Mordwinkin N., Plews J., Abilez O., Cui B., Gold J. & Wu J.
Nature methods, 2014
Cardiac differentiation strategy using a chemically defined medium consisting of just three components: the basal medium RPMI1640, l-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell. They first assessed chemically defined pluripotent culture on other defined matrices: rh E-cadherin, rh vitronectin, laminin-521, iMatrix-511, human fibronectin and a fibronectin mimetic. Laminin-based matrices resulted in higher growth rates compared to the vitronectin peptide. Fibronectin-based matrices did not support pluripotent growth. All five suitable matrices supported efficient differentiation in CDM3 but only the laminin-based matrices maintained long-term adhesion (>15 d) during CDM3 cardiac differentiation. The authors state that laminin-521 is an optimal matrix for chemically defined differentiation of human iPSC to cardiomyocytes but they still performed all subsequent characterization on the vitronectin peptide due to cost awareness.
Minami I., Yamada K., Otsuji T.G., Yamamoto T., Shen Y., Otsuka S., Kadota S., Morone N., Barve M., Asai Y., Tenkova-Heuser T., Heuser J.E., Uesugi M., Aiba K., and Nakatsuji N.
Cell Reports, 2012
In this study, they report of a small molecule that promote cardiac differentiation of hPSCs. By using this chemical, a xeno-free and cytokine-free cardiac differentiation protocol was achieved. In a part of the study cardiac differentiation on surface coating with gelatin or human laminin-211. Cell attachment on gelatin or laminin-211 is essential for efficient differentiation, suggesting that mechanotransduction or integrin signaling from interaction with these substrates might be important. iPSCs were differentiated into cardiac colonies on human laminin 211-coated dishes in cytokine-free and xeno-free defined medium containing HAS.
Kuroda T., Yasuda S., Matsuyama S., Tano K., Kusakawa S., Sawa Y., Kawamata S., Sato Y.
Regenerative Therapy, 2015
Human iPSCs were maintained on laminin-521 in Essential 8 medium. Cardiac differentiation on laminin-521 and Matrigel. Adult human cardiomyocytes (Promocell, Heidelberg) were cultured on laminin-211 in the Promocell myocyte growth medium. The authors have established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). LIN28 was the most sensitive marker of residual undifferentiated cells in hiPSC-derived cardiomyocytes but also in other tissues such as liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. Hence, the LIN28/ddPCR method is applicable to the quality control of hiPSC-derived cell therapy products.
Ja K.P., Miao Q., Zhen Tee N.G., Lim S.Y., Nandihalli M, Ramachandra C.J.A, Mehta A, Shim W. J
Cell Mol Med. 2015
Here they investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in immune deficient mice. iPSC cultured on Matrigel and differentiated in EB structures. Differences in integrin and laminin expression between cardiac progenitors and cardiomyocytes were observed. The a6 integrin were higher expressed in progenitors and integrin a1, a2, a3, a7 and b1 higher expression in cardiomyocytes. They observed a distinct swish in expression profile of laminin subunits during cardiac differentiation with laminin-411/421 pre-dominantly expressed early in progenitors and laminin-211/221 expressed later in cardiomyocytes. Improvements of myocardial function in the progenitor group corresponded to increased vascularization and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone. Laminin-221/211-expressing cardiomyocytes only retained and engrafted around myofibres in the peri-infarct region.
Penton C.M., Badarinarayana V., Prisco J., Powers E., Pincus M., Allen R.E., August P.R.
Skeletal Muscle, 2016
Here, the authors comprehensively examine the effect of physiologically relevant laminins, laminin-211 and laminin-521, compared to traditionally utilized ECMs (e.g., laminin-111, gelatin, and Matrigel) to assess their capacity to propagate and preserve myogenic differentiation potential. The results demonstrate laminin-521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Laminin-521 also provides more consistent and reliable differentiation over long-term culture. Laminin-521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin-211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed well in short-term mouse studies but showed high amounts of variability following long-term expansion.
Månsson-Broberg A., Rodin S., Bulatovic I., Ibarra C., Löfling M., Genead R., Wärdell E., Felldin U., Granath C., Alici E., Le Blanc K., Smith E.C.I., Salasova A., Westgren M., Sundström E., Uhlen P., Arenas E., Sylven C., Tryggvason K., Corbascio M., Simonson O.E., Österholm C., Grinnemo K-H.
Stem cell reports, 2016
The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) is investigated. Human fetal heart show immunohistochemical staining of laminin chains a2, a5, and g1. Cardiac MSCs were reproducibly recovered from the adherent cell fraction from first trimester human fetal hearts by using defined culturing conditions, including the use of specific laminin (LN) isoforms, in combination with canonical Wnt/b-catenin stimulation. The fetal cardiac MSCs stably expressed early cardiovascular markers, where subpopulations also expressed the stemness and potential cardiovascular progenitor markers. The cells expanded rapidly without changing their phenotype during the culturing process. The fetal cardiac MSCs were multipotent and could be differentiated into elongated, striated, and spontaneously beating cardiomyocytes, endothelial cells, and smooth muscle cell-like cells, also when using defined laminins as substrata. The MSCs could be differentiated into spontaneously beating, striated TnT+ cardiomyocytes, although it was only a small proportion of the cardiac MSCs that demonstrated this ability. Excluding either Wnt3a or laminin-based matrix from the culture protocol affected either cell growth or the gene expression of cardiac progenitor markers. By using LN-211 alone, the main component of the basement membrane surrounding adult cardiac and skeletal muscle fibers in situ, the cardiac MSCs could be differentiated into the cardiomyocyte lineage, with a 150 times increase of TNNT2 expression and a concomitant upregulation of NKX2.5. In addition, culture on LN-521/411, which are present in the basal membranes of vessels supported differentiation toward endothelial cells expressing CD31.