The Proceedings of the National Academies of Science (PNAS) has published the article that was the subject of this blog last week, and which claims that researchers at the University of Texas at Houston have produced the "first transplantable source of lung epithelial cells." There is no evidence that these cells are "transplantable," and they are definitely not the first team to produce Alveolar Type II ("ATII") lung cells from more primitive stem cells. The article does an excellent, if technical, job of explaining the importance of ATII lung epithelial cells:
The alveolar epithelium covers [approximately] 99%of the internal surface area of the lung and is composed of two major cell types, the alveolar type I (ATI) cell and the alveolar type II (ATII) cell. ATI cells are large flat cells through which exchange of CO2/O2 takes place. They cover [approximately] 95% 0f the alveolar surface and comprise [approximately] 40% of the alveolar epithelium and 8% of the peripheral lung cells. In contrast, ATII cells are small, cuboidal cells that cover [approximately] 5% of the alveolar surface and comprise 60% of the alveolar epithelium and 15% of the peripheral lung cells. They are characterized by the unique ability to synthesize and secrete surfactant protein C (SPC) and by the distinct morphological appearance of inclusion bodies, known as lamellar bodies. Important functions of ATII cells are (i) to synthesize, store, and secrete surfactant, which reduces surface tension, preventing collapse of the alveolus; (ii) to transport ions from the alveolar fluid into the interstitium, thereby minimizing alveolar fluid and maximizing gas exchange; (iii) to serve as progenitor cells for ATI cells, which is particularly important during reepithelialization of the alveolus after lung injury; and (iv) to provide pulmonary host defense by synthesizing and secreting several complement proteins including C3 and C5 (1–3) as well as numerous cytokines and interleukins that modulate lymphocyte, macrophage, and neutrophil functions (4). Severe pulmonary diseases can be caused by deficiencies or genetic mutations in proteins synthesized by ATII cells that are important in maintaining normal lung homeostasis. For example, complete deficiency of surfactant protein B (SPB) is caused by genetic mutations in the SPB gene. This deficiency results in impaired pulmonary surfactant composition and function and is a major cause of fatal neonatal respiratory disease (5, 6). In addition, ATII cells synthesize and secrete the serine protease inhibitor alpha-1-antitrypsin (alpha-1 AT) which also plays a key role in alveolar homeostasis by regulating protease imbalance and adjusting fluid clearance (7, 8), the importance of which is supported by the association of alpha-1 AT deficiency with the development of pulmonary emphysema (9). Cystic fibrosis is thought to be primarily a disease of the upper airway and submucosal epithelia and is caused by mutations in the cystic fibrosis transmembrane conductance receptor (CFTR) (10). CFTR is an important regulator of Cl and liquid transport in the lung (11–14) and is functionally expressed by human ATII cells, strongly suggesting a critical role for CFTR in regulating ion and fluid transport in the lung alveolus in addition to the upper airway (13).(The numbers in parentheses refer to footnotes. Also, I had to change some of the characters to words: "alpha" and "approximately")
The PNAS report and UT Houston's Press Release do not contain any note about the earlier umbilical cord blood stem cell research, although the latter was published on line and in print in Cytology, at least 2 weeks before the initial submission of the PNAS article.
Both research teams report that they followed the techniques developed and reported in the lab of another researcher, Samadikuchaksaraei, in growing, multiplying and guiding the differentiation of their primitive cells toward the more specialized lung cells that were desired. Both report the successful production of ATII lung cells, as demonstrated by the way the cells look and by demonstrating the production of Surfactant Protein C - which, in human development is only found in mature ATII cells after the unborn (or premature) child has reached 36 weeks of gestation.
The Houston team claims that one reason their process is superior to the earlier Embryonic Stem Cell research is that they were able to produce mature cells in 10 days, while Samadikuchaksaraei's team took 15 days. If the ability to produce the cells in what Wetsel, et. al., describe as a "timely manner," then it is important to note that the Minnesota team produced their mature ATII cells in 3 to 8 days.
The Houston team also claims that is possible that their new cell lines might one day be transplanted, although there has never been any research reporting the successful transplantation of epithelial cells into the lungs. Another problem is that the cells were guided to change by "transfection" with a segment of DNA that is inserted into the genes of the cells, using a retrovirus. Any use of these cells, even if anyone ever proves that we can transplant cells into the lung and cure a disease, will be complicated by years of research to prove that the gene therapy that produced these cells is safe and stable in the lungs of the patients. The authors do not give us any references to support this hypothesis.
From the Discussion section of the PNAS article:
"Lung injury due to chronic pulmonary diseases, such as chronic
obstructive pulmonary disease and asthma, and inherited genetic disorders, such as cystic fibrosis and 1-AT deficiency are leading causes of morbidity and mortality worldwide. Cystic
fibrosis and 1-AT deficiency are two of the most common inherited genetic defects affecting Caucasians. In addition, SPB deficiency is a major cause of respiratory disease and fatality in neonates. All three of these diseases are caused by single-gene defects and therefore have been logical candidates for gene therapy. However, efficient vector delivery and sufficient transgene expression needed for therapeutic benefit have remained elusive. Recent research advances indicate that gene delivery via transplantation of cells derived from human stem cells may provide an attractive alternative to viral or liposome vector based gene therapies. Moreover, transplantation of cells derived from human stem cells may prove ideal for the repair and regeneration of injured lung tissue.
Because of its ability to proliferate as well as to differentiate into ATI cells, the ATII cell is an excellent choice of lung cell for possible therapeutic use in gene delivery and repair of the alveolus.
. . . The use of ES cells as a source of transplantable cells in the lung alveolus will require the generation of significant quantities of highly pure ATII cells. To achieve this goal, we chose to genetically modify hES cells so that resulting differentiated ATII cells could be enriched through antibiotic selection. Our approach was to establish a stable transfected hES cell line containing a single copy of the human SPC promoter-Neor fusion gene. When subjected to differentiation in vitro, it was hypothesized that ATII cells derived from this genetically modified hES cell line (SPCP/NEO.74) would express the Neor gene and would therefore survive G418 antibiotic selection, whereas, all of the other differentiated cell lineages as well as the pluripotent cells would be eliminated by G418 selection. Immunocytochemical and flow cytometric analysis of the surviving G418-selected cells supported this hypothesis, indicating that this genetic selection approach resulted in an enrichment of hES-ATII cells to 99% when cultured on Matrigel-coated plates. Our protocol reproducibly produced from each 10-cm culture dish 106 essentially pure ATII cells within 15 days of differentiation. These differentiated ATII cells survive for at least 2 days in culture in the absence of G418 and will provide in a timely manner sufficient numbers of pure ATII cells for future transplantation investigations."
The abstracts are available on line for free, but the actual articles are available only by subscription or by paying for temporary access:
From Proceedings of the National Academies of Science, published online March 2, 2007
"A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells"
Dachun Wang, David L. Haviland, Alan R. Burns, Eva Zsigmond, and Rick A. Wetsel. Research Center for Immunology and Autoimmune Diseases and Laboratory for Developmental Biology, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center; Department of Biochemistry and Molecular Biology, University of Texas Medical School; and Cardiovascular Sciences Section, Department of Medicine, Baylor College of Medicine. Communicated by C. Thomas Caskey, University of Texas Health Science Center, Houston, TX, January 4, 2007 (received for review November 22, 2006)
Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.
Keywords: complement , differentiation, surfactant proteins, alpha-1-antitrypsin, cystic fibrosis transmembrane conductance receptor
"Differentiation of umbilical cord blood-derived multilineage progenitor cells into respiratory epithelial cells."
MJ Berger, SD Adams, BM Tigges, SL Sprague, X-J Wang, DP Collins and DH McKenna, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota, USA, Clinical Cell Therapy Laboratory, University of Minnesota Medical Center, Minneapolis, Minnesota, USA, and BioE Inc., Saint Paul, Minnesota, USA
Background - Umbilical cord blood (UCB) has been examined for the presence of stem cells capable of differentiating into cell types of all three embryonic layers (i.e. endo-, ecto- and mesoderm). The few groups reporting success have typically confirmed endodermal potential using hepatic differentiation. We report differentiation of human UCB-derived multipotent stem cells, termed multilineage progenitor cells (MLPC), into respiratory epithelial cells (i.e. type II alveolar cells).
Methods - Using a cell separation medium (PrepaCyte-MLPC; BioE Inc.) and plastic adherence, MLPC were isolated from four of 16 UCB units (American Red Cross) and expanded. Cultures were grown to 80% confluence in mesenchymal stromal cell growth medium (MSCGM; Cambrex BioScience) prior to addition of small airway growth medium (SAGM; Cambrex BioScience), an airway maintenance medium. Following a 3 - 8 day culture, cells were characterized by light microscopy, transmission electron microscopy, immunofluorescence and reverse transcriptase (RT)-PCR.
Results - MLPC were successfully differentiated into type II alveolar cells (four of four mixed lines; two of two clonal lines). Differentiated cells were characterized by epithelioid morphology with lamellar bodies. Both immunofluorescence and RT-PCR confirmed the presence of surfactant protein C, a protein highly specific for type II cells.
Discussion - MLPC were isolated, expanded and then differentiated into respiratory epithelial cells using an off-the-shelf medium designed for maintenance of fully differentiated respiratory epithelial cells. To the best of our knowledge, this is the first time human non-embryonic multipotent stem cells have been differentiated into type II alveolar cells. Further studies to evaluate the possibilities for both research and therapeutic applications are necessary.
Keywords - endodermal differentiation, respiratory epithelium, stem cells, umbilical cord blood.
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