miércoles, 25 de marzo de 2015

Scientists coax stem cells to form 3-D mini lungs

University of Michigan Health System
Scientists have coaxed stem cells to grow the first three-dimensional mini lungs. Previous research has focused on deriving lung tissue from flat cell systems or growing cells onto scaffolds made from donated organs.

In a study published in the online journal eLife the multi-institution team defined the system for generating the self-organizing human lung organoids, 3D structures that mimic the structure and complexity of human lungs.

Figure 1. Generation of three-dimensional ventral anterior foregut spheroids from endoderm monolayers.
(A) hESCs were differentiated into foregut endoderm by treating cells with 4 days of Activin A (ACTA) followed by 4 days of NOG+SB. (B) Foregut endoderm (NOG+SB) had high expression of the foregut marker SOX2 while the hindgut marker CDX2 was significantly reduced compared to untreated endoderm controls (End). NOG+SB monolayers had high expression of ventral anterior foregut genes NKX2.1 and PAX8 while the posterior foregut marker PDX1 was reduced. The foregut marker HHEX is expressed in the developing liver, biliary system, and thyroid and remained unchanged. (C) The majority of cells in NOG+SB treated cultures were SOX2 positive (green) compared to the control, in which only scattered clusters of cells were SOX2 positive. The scale bar represents 200 µm. (D) hESCs were differentiated into foregut spheroids by treating cells with 4 days of ACTA and then additional 4–6 days of NOG+SB+FGF4+Ch. Representative images of a spheroid in a matrigel droplet are shown as a whole mount image. Scale bar represents 100 µm. (E) Foregut spheroids (NOG+SB+FGF4+Ch) had high expression of the foregut marker SOX2 while the hindgut marker CDX2 was significantly reduced compared to untreated endoderm control (End) (top panel). Spheroids had high expression of anterior foregut genes NKX2.1 and PAX8 while the posterior foregut marker PDX1 was reduced and HHEX was unchanged (bottom panel). *p < 0.05, error bars represent SEM. (F) The majority of cells in foregut spheroids are FOXA2+ (green, left panel) and SOX2+ (white, right panel) and ECAD+ (red, right panel). Scale bar represent 50 µm.

Figure 1—figure supplement 2.Foregut spheroids co-express endoderm and lung specific markers.
(A) NOG/SB/FGF4/Ch spheroids have weak NKX2.1 (green) expression which co-expresses with endoderm marker FOXA2 (red). (B) The majority of cells in the spheroid express SOX2 (green) and co-stain with FOXA2 (red). Scale bars represent 50 µM.
Figure 1—figure supplement 3. Foregut spheroids consist of both epithelial and mesenchymal cells.
NOG/SB/FGF4/Ch spheroids have a minor population of Vimentin (VIM, white) positive mesenchymal cells, while the majority of cells are epithelial and express ECAD (red). Scale bar represents 50 µM.
Figure 1—figure supplement 4. NOG+SB+FGF4+Ch spheroids do not express neural markers.
hESCs were differentiated into endoderm by treating with 4 days of ActivinA (ACTA) and spheroids were generated with an additional 4 days of NOG+SB+FGF4+Ch. Neural cultures were not treated with ACTA, but were treated with NOG+SB for 8 days. Compared to foregut spheroids (NOG+SB+FGF4+Ch), NOG+SB neural cultures had a significant increase in neural markers NESTIN, SOX1, and PAX6 and significant decrease in FOXA2, which is highly expressed in endoderm. *p < 0.05, error bars represent SEM.

"These mini lungs can mimic the responses of real tissues and will be a good model to study how organs form, change with disease, and how they might respond to new drugs," says senior study author Jason R. Spence, Ph.D., assistant professor of internal medicine and cell and developmental biology at the University of Michigan Medical School.

The scientists succeeded in growing structures resembling both the large airways known as bronchi and small lung sacs called alveoli.

Since the mini lung structures were developed in a dish, they lack several components of the human lung, including blood vessels, which are a critical component of gas exchange during breathing.
Still, the organoids may serve as a discovery tool for researchers as they churn basic science ideas into clinical innovations. A practical solution lies in using the 3-D structures as a next step from, or complement to, animal research.

Cell behavior has traditionally been studied in the lab in 2-D situations where cells are grown in thin layers on cell-culture dishes. But most cells in the body exist in a three-dimensional environment as part of complex tissues and organs.
Researchers have been attempting to re-create these environments in the lab, successfully generating organoids that serve as models of the stomach, brain, liver and human intestine.

The advantage of growing 3-D structures of lung tissue, Spence says, is that their organization bears greater similarity to the human lung.

How to make a human lung in a dish
To make these lung organoids, researchers at the U-M's Spence Lab and colleagues from the University of California, San Francisco; Cincinnati Children's Hospital Medical Center; Seattle Children's Hospital and University of Washington, Seattle manipulated several of the signaling pathways that control the formation of organs.

First, stem cells -- the body's master cells -- were instructed to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and several other internal organs.

Scientists activated two important development pathways that are known to make endoderm form three-dimensional tissue. By inhibiting two other key development pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos.
In the lab, this early lung-like tissue spontaneously formed three-dimensional spherical structures as it developed. The next challenge was to make these structures expand and develop into lung tissue. To do this, the team exposed the cells to additional proteins that are involved in lung development.

The resulting lung organoids survived in the lab for over 100 days.

"We expected different cells types to form, but their organization into structures resembling human airways was a very exciting result," says lead study author Briana Dye, a graduate student in the U-M Department of Cell and Developmental Biology.

Other sourcesRecipe For How To Grow A Human Lung
from Scientific Blogging
ORIGINAL: eScience News
Source: University of Michigan Health System
March 25, 2015

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