Efficient Derivation of Alveolar Type II Cells from Embryonic Stem Cells forIn VivoApplication

Abstract

In the present study, mouse embryonic stem cells (ESCs) were differentiated into alveolar epithelial type II (AEII) cells for endotracheal injection. These enriched lung-like populations expressed lung epithelial markers SP-A, SP-B, SP-C, and CC10. First we show that rapid differentiation of ESCs requires a dissociated seeding method instead of an embryoid body culture method. We then investigated a two-step differentiation of ESCs into definitive endoderm by activin or A549-conditioned medium as a precursor to lung epithelial cells. When conditioned medium from A549 cells was used to derive endoderm, yield was increased above that of activin alone. Further studies showed that Wnt3a may be one of the secreted factors produced by A549 cells and promotes definitive endoderm differentiation, in part, through suppression of primitive endoderm. Activin and Wnt3a together at appropriate doses with dissociated cell seeding promoted greater endoderm yield than activin alone. Next, fibroblast growth factor 2 was shown to induce a dose-dependent expression of SPC, and these cells contained lamellar bodies characteristic of mature AEII cells from ESC-derived endoderm. Finally, ES-derived lung cells were endotracheally injected into preterm mice with evidence of AEII distribution within the lung parenchyma. This study concludes that a recapitulation of development may enhance derivation of an enriched population of lung-like cells for use in cell-based therapy.

Differentiation of Mouse Induced Pluripotent Stem Cells Into Alveolar Epithelial Cells In Vitro for Use In Vivo

“…Mouse iPSCs were induced to differentiate into AEC II-like cells using the dissociated seeding method for murine ESCs described previously, with slight modifications [10]. As shown in Fig.…”

“…Embryonic stem cells (ESCs) are self-renewing pluripotent cells that can differentiate into alveolar epithelial cells in vitro [5][6][7][8][9][10][11][12][13][14][15][16]. The problems of immune rejection and ethical issues restrict clinical application of ESCs.…”

“…We used a previously reported two-step protocol to differentiate iPSCs into AECs. Although most ESC differentiation protocols have used the conventional embryoid body method [9], we achieved iPSC differentiation using a dissociated low-density seeding method that is considered more rapid and effective [10]. We tested six medium combinations to identify the most efficient protocol and successfully differentiated mouse iPSCs into AEC-like cells.…”

EngineeringDe NovoAssembly of Fetal Pulmonary Organoids

“…2 Currently, extensive research efforts are aimed at engineering whole lungs for implantation, with the majority of attention focused on the use of decellularized cadaveric lungs recellularized with pulmonary derivatives of various stem/progenitor cell sources. [3][4][5] Over the past few years, significant progress has been made in understanding specific pathways that drive the directed differentiation of anterior foregut endoderm and lung epithelia from murine and human embryonic stem cells [6][7][8][9][10] and human induced pluripotent stem cells. 9,[11][12][13] In addition, there have been several recent reports that identify diverse resident lung stem/progenitor cells in humans 14,15 ; however, very little is known about the growth factor and extracellular matrix (ECM) cues required to drive de novo tissue formation by competent lung progenitor cells in vitro.…”

Directed Differentiation of Mesendoderm Derivatives from Embryonic Stem Cells

“…The idea of transitioning lung epithelia through known normal developmental stages, which has been very successful for hepatocytes, may also be applied to lung. For example, initial generation of anterior endoderm using ActivinA, coupled with Wnt3a to suppress primitive endoderm, was successful using a subsequent step of FGF2 induction, to generate SPC-positive cells characteristic of type II alveolar epithelium (Roszell et al, 2009). For the generation of human lung epithelial cells from human ES cells there is to date only a single published protocol (Samadikuchaksaraei et al, 2006).…”