Cynata’s MSC Technology Produces Significant Relief of Asthma in Preclinical Study


An Australian stem cell company called Cynata Therapeutics Limited is in the process of developing a therapeutic stem cell platform technology that they called “Cymerus.” The idea for Cymerus originated at the University of Wisconsin-Madison, but Cymerus would generate a protocol by which clinical laboratories could produce very immature mesenchymal stem cells from induced pluripotent stem cells. Such cells would be personalized for patients and their needs, and Cynata’s goal is to produce a platform that is economically feasible and relatively fast so that patients can receive infusions of the cells they so badly need in a timely fashion. These are very ambitious goals to say the least, but Cynata has been hacking away at this problem for some time, and we certainly wish them the best.

Cynata has recently released some very encouraging data in which their personalized mesenchymal stem cells were used to treat laboratory animals with a laboratory-induces form of asthma. Briefly, female mice (BALB/c mice for those who are interested) were injected with a yolk-protein called “ovalbumin.” Ovalbumin is a protein found in egg whites, and because it is an egg-specific protein, mice do not have it and their immune systems have never seen it before. Such an injection causes the mice to mount an immune response to the ovalbumin, and these mice are then administered aerosolized ovalbumin by means of a nebulizer. This causes the animals to develop a rather severe asthmatic attack against ovalbumin.

In this study, Cynata scientists and their collaborators used 48 mice that were divided into six different groups. The first group was untreated animals that did not suffer from ovalbumin asthma. The second group contained eight animals that had no asthma but were treated intravenously with one million mesenchymal stem cells. The third group also had no asthma, but were treated with an intranasal infusions of one million mesenchymal stem cells. The fourth group contain eight asthmatic animals that were untreated during the course of the experiment. The fifth group contain eight asthmatic animals that were treated intravenously with one million mesenchymal stem cells. The final group contained eight asthmatic animals that were treated with intranasal infusions of one million mesenchymal stem cells. As a note, all animals that were treated mesenchymal stem cells were treated three times. So-called airway hyperresponsiveness (AHR) is a measure of the sensitivity and irritability of the bronchial tissues. AHR is an important measure of the tendency of the lungs to undergo constriction during an asthma attack and AHR is usually measured by administering a drug that can cause bronchoconstriction. The greater the degree of bronchoconstriction in such an experiment is indicative of great AHR. The successful treatment of asthma results in reduction in AHR.

The results of this experiment were wonderfully successful. Exposing mice to the ovalbumin caused them to exhibit significantly increased AHR. However, intravenous administration of Cynata’s MSCs in asthmatic animals caused a statistically significant (60-70%) decrease in AHR compared to untreated, sensitized animals. Additionally, intranasal administration of Cynata’s MSCs completely normalized AHR. The AHR in these asthmatic mice was brought down to a level that was largely the same as the non-asthmatic mice. Also, importantly, no adverse side effects were observed during the study.

This study was conducted under the supervision of Associate Professor Chrishan Samuel and Dr. Simon Royce from the Department of Pharmacology at Monash University, Melbourne, Australia. Because the features of this model asthma system closely resemble the clinical manifestations of asthma in humans, these results provide excellent evidence that such a treatment stands a chance of working in human patients.

“We are very excited by these results, which indicate that Cymerus™ MSCs could have a profound effect in the treatment of asthma. This is a debilitating condition, which affects about 10% of the population, resulting in close to 40,000 hospitalizations and several hundred deaths each year, in Australia alone,” said Cynata Vice President of Product Development, Dr. Kilian Kelly. “Although a number of drugs are approved for the treatment of asthma, studies have shown that conventional treatments result in as few as 5% of asthma patients achieving full control of their condition. Consequently, there is a widely recognized need for novel treatments that address – and potentially eliminate – the underlying disease”, added Dr. Kelly.

“This study has clearly demonstrated that Cynata’s MSCs have a dramatic effect on AHR in our model, particularly when directly administered into the allergic lung. We look forward to continuing our analysis of the effects of these unique cells on markers of inflammation and airway remodeling, and we are optimistic of building on the very positive data we have generated so far,” said Associate Professor Samuel.

Asthma is a condition characterized by the inflammation, narrowing, and swelling of the airways, accompanied by excessive mucous production that makes it difficult to breathe. According to the Global Asthma Network, asthma affects over 330 million people globally. Cynata had partnered with Monash University to examine the potential of its Cymerus technology as an alternate treatment for asthma sufferers.

Cymerus™ makes us of induced pluripotent stem cells (iPSCs) that are then differentiated into a specific type of mesenchymal stem cell precursor known as a “mesenchymoangioblast” or MCA. Cymerus potentially provides a source of MSCs that can be made for so-called “off-the-shelf” therapeutic uses.

Fat-Based Mesenchymal Stem Cell-Seeded Matrix Heals Bronchopleural Fistula in Female Cancer Patient


Bronchopleural fistulae, mercifully abbreviated as BPF, refers to an opening or hole in the respiratory tree that causes continuity between the pleural space that surrounds the lungs and the bronchial tree. BPH is a highly feared complication of surgery on the respiratory system.

BPH can complicate surgical resection of the pulmonary system. Patients with lung cancers may require lung resection in order to remove tumorous lung tissue. The rate of BPH incidence after lung surgery varies widely, with reported incidences ranging from 1.5 to 28%. Necrosis or death of lung tissue as a result of infection can also cause BPH, as can tuberculosis. Chemotherapy or radiation therapy for lung cancers can also result in BPF. Finally, BPF may caused by persistent spontaneous pneumothorax, which refers to an abnormal build up of air or other gases in the pleural space, which causes an uncoupling of the lung from the chest wall.

To date, treatment for BPF is only partially effectively. The main treatment includes surgery, but the rate of recurrence of the fistulae remains rather high as do the rate of mortality. Can stem cells show us a better way?

Perhaps they can. Dennis A. Wigle, a surgeon at Mayo Clinic, and his collaborators used a synthetic bioabsorbable matrix seeded with the patients one fat-based mesenchymal stem cells to heal a BPF in a 63-yr old woman. Mind you, this is a case study (the lowest quality clinical evidence) and not a controlled study,. However, the success of this case study is at least suggestive that such an approach might prove useful for patients who suffer from BPFs.

Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.
Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.

A 63-yr old woman who had surgical resection of the lung in order to treat her lung cancer had, as a consequence of her surgery, a BPF. Some 30 different surgical attempts were made to repair the BPF, but all of them failed. The woman’s health declined and her medical team started to think of alternative treatments.

Fortunately, Mayo Clinic has been participating in an ongoing clinical trial to use fat-based mesenchymal stem cells to treat anal fistulae in Crohn’s disease patients. Therefore Dr. Wigle and his team considered using the protocol utilized with Crohn’s patients to repair this woman’s BPF.

Fat biopsies were taken from the patient and the fat was washed, minced, digested with enzymes, and then grown in special culture media. The adipose tissue-derived mesenchymal stem cells (AD-MSCs) grew and were isolated, characterized and shown to be MSCs.

These cells were then seeded on a matrix of synthetic bioabsorbable poly(glycolide-trimethylene carbonate) copolymer and then placed in a bioreactor to grow. After about 4 days, the matrix was flush with AD-MSCs, and this cell-seeded patch was then used in a subsequent surgery to seal the opening in the respiratory tree. This time the surgery worked. The patient was discharged 25 days after the surgery and sent home.

MRIs of the respiratory system showed that the BPF had indeed closed and properly resolved.

Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.
Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.

This case study might confirm what was previously observed in large animal studies by Petrella and others, namely that AD-MSCs can be used to heal BPF. Petrella and others theorized that implanted MSCs induce the proliferation of fibroblasts that then deposit collagen, which seals the BPF (see Ann Thorac Surg 97:480483.  Alternatively, AD-MSCs might differentiate into cell types  required for regeneration of the airways (Dominici M, and others, Cytotherapy 8:315317).  Either way, this paper seems to suggest that AD-MSCs can be isolated from a patient’s fat (even a very sick patient like this one) without incident and used for tissue engineering applications that can repair very serious wound like BPF. 

This paper was published in: Johnathon M., Aho, et Al., “Closure of a Recurrent Bronchopleural Fistula Using a Matrix Seeded With Patient-Derived Mesenchymal Stem Cells.” Stem Cells Trans Med October 2016 vol. 5 no. 10 1375-1379. 

New Stem Cell Treatment for Bronchopleural Fistulas


Mayo Clinic researchers have made history by using a patient’s own stem cells to heal an open wound on the upper chest of a patient that had been caused by postoperative complications of lung removal.

A hole in the chest that opens to the outside is called a bronchopleural fistulae. Such wounds are holes that lead from large airways in the lungs to the membrane that lines the lungs.

Unfortunately, present treatments for bronchopulmonary fistulae tend to be terribly successful and death from such injuries are all too common.

According to Dr. Dennis Wigle, a Mayo Clinch Researcher, “Current management is not reliably successful. After exhausting therapeutic options, and with declining health of the patient, we moved toward a new approach. The protocol and approach were based on an ongoing trial investigating this method to treat anal fistulas in Cohn’s disease”.

So Dr. Wigle and his colleagues harvested stem cells from the belly fat of their patient and seeded onto a bioabsorbable mesh that was surgically implanted at the site of the fistula.

Follow-up imaging of the patient showed that the fistula had closed and remained healed. More than a year-and-a-half later, the patient remains asymptomatic and has been able to resume activities of daily living.

In their paper, Wigle and others describe their patient, a 63-year-old female patient, who was referred to Mayo Clinic for treatment of a large bronchopleural fistula.

Because present therapies offer little relief, Wigle and his team turned to regenerative therapies in order to try a more innovative treatment.

“To our knowledge, this case represents the first in human report of surgically placed stem cells to repair a large, multiple recurrent bronchopleural fistula. The approach was well tolerated suggesting the potential for expanded use,” said Dr. Wigle.

While this procedure was successful in this case, it is unclear if this treatment was the main contributor to the healing of the wound. Since this is a single-patient case study and not a double blinded, placebo-controlled study, it is lower-quality evidence.

However, Wigle and others hope to further examine this technique, and in particular, the use a patient’s own stem cells, to treat fistulae in the respiratory system.

This case study was published in Stem Cells Translational Medicine, June 2016 DOI:10.5966/sctm.2016-0078.

Alveolar Macrophages Derived from Stem Cells Help Lung-Damaged Mice Recover and Survive from Airway Disease


Within the tiny alveolar sacs of our lungs is an immune cell that surveys and directs the immune response within the lung. This immune cell is called an “alveolar macrophage,” and this cell is an actively phagocytic cell. It gobbles up invading bacteria and foreign material in order to keep the lungs clean. When these cells work normally, they help our lungs function properly. When, however, they go rogue, they can fill the lungs with cells that clog the lungs and prevent you from breathing.

Alveolar Macrophages
Alveolar Macrophages

Certain diseases like chronic obstructive pulmonary disease, asthma, and lung fibrosis, have abnormal alveolar macrophages and no specific treatments can appropriately compensate for these abnormalities.

Since alveolar macrophages (AMs) can be made from pluripotent stem cells, perhaps transplanting exogenous AMs derived from pluripotent stem cells can clean up messy lungs.

Martin Post, from the University of Toronto, in Ontario, Canada, and his colleagues tested this very hypothesis in mice. Post and his coworkers differentiated mouse embryonic stem cells by using factor-defined media in order to generate embryonic macrophages that could be grown in culture. Then they conditioned their cells into an alveolar-like phenotype by treating them with the cytokine GM-CSF. The cells were surprisingly like normal AMs, at least in culture.

To test these cells in mice, Post and his group created mice that lacked the ADA (adenine deaminase) gene and these mice lacked proper AM activity and suffered chronic lung damage.

Next, Post’s team transplanted their embryonic stem cell-derived alveolar-like macrophages into the tracheas (windpipes) of these injured animals in order to view their therapeutic potential.

What Post and others saw truly amazed them. Not only was their differentiation protocol wonderfully efficient and adaptable to human pluripotent stem cells, but their PSC-derived macrophages essentially “walked and talked” like regular, normal AMs. These cells made all the right cell surface proteins to be identified as AMs and they engulfed bacteria and dying cells. In fact, they were better phagocytes than bone marrow-derived macrophages.

The implanted macrophages stayed in the airways of the recipient mice for at least 4 weeks, and were able to gobble up other types of rogue white blood cells (i.e., neutrophils) during acute lung injury. Thus, the implanted cells were able to protect the lung from further damage under conditions of lung injury. Additionally, the implanted AMs enhanced tissue repair in the lungs and promoted survival of these mice. Interestingly, the mice did not develop abnormal pathology or teratomas as a result of the implanted macrophages.

Thus, this work from Post and his colleagues shows that pluripotent stem cells are a viable source of therapeutically effective alveolar-like macrophages that can be implanted into the lungs and treat airway diseases. Further experiments in larger animals should prepare this strategy for clinical trials.

This study was published in the American Journal of Respiratory and Critical Care Medicine. published online 05 Jan 2016 as DOI: 10.1164/rccm.201509-1838OC.

Scientists Grow New Diaphragm Tissue In Laboratory Animals


The diaphragm is a parachute-shaped muscle that separates the thoracic cavity from the abdominopelvic cavity and facilitates breathing. Contraction of the diaphragm increases the volume of the lungs, thus dropping the pressure in the lungs below the pressure of the surrounding air and causing air to rush into the lungs (inhalation). Relaxation of the diaphragm decreases the volume of the lungs and increases the pressure in the lungs so that it exceeds the pressure of the air, and air leaves the lungs (exhalation). The diaphragm is also important for swallowing. One in 2,500 babies are born with malformations or perforations in their diaphragms, and this condition is usually fatal.

The usual treatment for this condition involves the construction of an artificial patch that properly covers the lesion, but has no ability to grow with the infant and is not composed of contractile tissue. Therefore, it does not assist in contraction of the diaphragm to assist in breathing.

A new study might change the prospects for these newborn babies. Tissue engineering teams from laboratories in Sweden, Russia and the United States have successfully grown new diaphragm tissue in rats by applying a mixture of stem cells embedded in a 3D scaffold made from donated diaphragm tissue. Transplantation of this stem cell/diaphragm matrix concoction into rats allowed the animals to regrow new diaphragm tissue that possessed the same biological characteristics as diaphragm muscle.
The techniques designed by this study might provide the means for repairing defective diaphragms or even hearts.

Doris Taylor, who serves as the director of regenerative medicine research at the Texas Heart Institute and participated in this revolutionary study, said: “So far, attempts to grow and transplant such new tissues have been conducted in the relatively simple organs of the bladder, windpipe and esophagus. The diaphragm, with its need for constant muscle contraction and relaxation puts complex demands on any 3D scaffold. Until now, no one knew whether it would be possible to engineer.”

Paolo Macchiarini, the director of the Advanced Center for Regenerative Medicine and senior scientist at Karolinska Institutet, who also participated in this study, said: “This bioengineered muscle tissue is a truly exciting step in our journey towards regenerating whole and complex organs. You can see the muscle contracting and doing its job as well as any naturally grown tissue.”

To make their tissue engineered diaphragms, the team used diaphragm tissue that had been taken from donor rats. They stripped these diaphragms of all their cells, but maintained all the connective tissue. This removed anything in these diaphragms that might cause the immune systems of recipient animals to reject the implanted tissue, while at the same time keeping all the things that give the diaphragm its shape and form. In the laboratory, the decellularized diaphragms had lost all their elasticity. However, once these diaphragm matrices were seeded with bone marrow-derived stem cells and transplanted into recipient laboratory animals, the diaphragm scaffolds began to function as well as normal, undamaged diaphragms.

If this new technique can be successfully adapted to human patients, it could replace the damaged diaphragm tissue of the patient with tissue that would constantly contract and grow with the child. Additionally, the diaphragm could be repaired by utilizing a child’s own stem cells. As a bonus, this technique might also provide a new way to

Next, the team must test this technique on larger animals before it can be tested in a human clinical trial.

The study was published in the journal Biomaterials.

Lung Spheroidal Cells Superior to Mesenchymal Stem Cells When Treating Pulmonary Fibrosis in Mice


Lung diseases are no fun for anyone. The constant feeling of suffocation, withering weakness, and significant limitations on human activity are indicative of a loss of lung capacity. Chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis are among the top five causes of mortality, according to the World Health Organization (Cottin Eur Respir Rev 22:2632).  Regenerating damaged lungs, therefore, represent one of the Holy Grails of regenerative medicine.

Animal studies have used infusions of mesenchymal stem cells (MSCs) from isolated from human bone marrow, adipose tissue, placental tissue, or cord blood to treat animals with various types of lung disease (Moodley Y, et al.Am J Pathol 175:303313; Ortiz LA, et al. Proc Natl Acad Sci USA 104:1100211007; Ortiz LA, et al. Proc Natl Acad Sci USA 100:84078411). Also, a Phase I clinical trial has assessed the safety of fat-based MSCs as a treatment for lung damage in human patients. Because this study was only designed to test the safety of this procedure, little to nothing can be said of the efficacy of this test (Tzouvelekis A, J Transl Med. 2013 Jul 15;11:171).

Recently, several laboratories have identified resident stem cells in the lung and some researchers have even managed to isolate them and growth them in culture (Desai TJ, et al., (2014) Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507:190–194; Kajstura J, et al., (2011) Evidence for human lung stem cells. N Engl J Med 364:1795–1806; Kim CF, et al., (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835; Wansleeben C, et al. (2013) Stem cells of the adult lung: Their development and role in homeostasis, regeneration, and disease. Wiley Interdiscip Rev Dev Biol 2:131–148; Barkauskas CE, et al. (2013) Type 2 alveolar cells are stem cells in adult lung. J Clin Invest 123:3025–3036; Hogan BL, et al. (2014) Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15:123–138). Could such cells work better than MSCs?

At this point it is difficult to say, since there are a fair amount known about isolating and growing MSCs in culture, but there is relatively little known about resident lung stem cell populations. However, a new paper might change that feature of the debate.

Ke Cheng and his colleagues from North Carolina State University have developed a rapid, reproducible, and scalable method to generate clinically applicable amounts of resident lung progenitor cells. This technique capitalizes on the “Spheroid method” used for tumor cells. Spheroids are three-dimensional structures that grow as small balls of cells in culture. Cheng and others applied the spheroid culture method to lung cells with great success.

Lung cells were acquired from lung biopsies taken from human patients. The tissues were appropriately minced, treated with enzymes to disintegrate the structural components, and then grown in culture. The cells that grew were eventually seeded onto a special culture system for growing spheroids. In this culture system, spheroids formed, consisting of internal clumps of lung progenitor cells surrounded by a shell of stroma-like cells. These cells expressed the cadre of genes you would expect them to. Cheng and his coworkers called these cells “lung spheroid cells” or LSCs.

Generation of lung spheroids and lung spheroid cells. (A): Schematic showing the protocol to grow lung spheroids and lung spheroid cells. (B-I): Edge of lung tissue explants with outgrowth cells becoming confluent and ready to harvest. (B-II): Lung spheroids formed from outgrowth cells in suspension culture. (B-III): Plated lung spheroids onto fibronectin-coated surfaces to generate lung spheroid cells. (B-IV): Expansion of LSCs in suspension cultures. (C): Cumulative doubling for LSCs from three different donors. (D): Immunocytochemistry on lung spheroids. Scale bars = 50 µm. Abbreviations: LSCs, lung spheroid cells; PF, pulmonary fibrosis; SCID, severe combined immunodeficiency.
Generation of lung spheroids and lung spheroid cells. (A): Schematic showing the protocol to grow lung spheroids and lung spheroid cells. (B-I): Edge of lung tissue explants with outgrowth cells becoming confluent and ready to harvest. (B-II): Lung spheroids formed from outgrowth cells in suspension culture. (B-III): Plated lung spheroids onto fibronectin-coated surfaces to generate lung spheroid cells. (B-IV): Expansion of LSCs in suspension cultures. (C): Cumulative doubling for LSCs from three different donors. (D): Immunocytochemistry on lung spheroids. Scale bars = 50 µm. Abbreviations: LSCs, lung spheroid cells; PF, pulmonary fibrosis; SCID, severe combined immunodeficiency.

When Cheng and his group implanted LSCs into Matrigel, the cell appropriately differentiated into lung-specific cell types and formed structures that greatly resembled the tiny air sacs in lungs, known as alveoli. These structures, based on their expression patterns of particular genes are where specific proteins were found in the cells, seemed for a mature lung structure. Interestingly, when the culture medium that had been used to grow the LSCs (conditioned media) was given to other cultured cells, it promoted survival or proliferation of human lung epithelial cells and tube formation of human endothelial cells on Matrigel.

In vitro differentiation and paracrine assays of lung spheroid cells. (A): LSCs grown on Matrigel and displaying alveoli-like structures (inset). (B): LSCs grown on Matrigel expressed aquaporin 5 (red). (C): Human lung epithelial cells cultured in control media and LSC-CM and stained for live (green)/dead (red) assay. (D): HUVEC tube formation assay on Matrigel surface in control or conditioned media from LSCs. Data are presented as mean ± SD. All experiments were run in triplicate, unless noted otherwise. Scale bars = 50 µm. ∗, p < .05 compared with the control media group. (E): Representative antibody array images showing the proteins presenting in the CM from LSCs and NHDF cells. Abbreviations: AQ5, aquaporin 5; BDNF, brain-derived neurotrophic factor; CM, conditioned media; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GRO, growth-regulated protein; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; LSCs, lung spheroid cells; POS, positive; NHDF, normal human dermal fibroblast cell.
In vitro differentiation and paracrine assays of lung spheroid cells. (A): LSCs grown on Matrigel and displaying alveoli-like structures (inset). (B): LSCs grown on Matrigel expressed aquaporin 5 (red). (C): Human lung epithelial cells cultured in control media and LSC-CM and stained for live (green)/dead (red) assay. (D): HUVEC tube formation assay on Matrigel surface in control or conditioned media from LSCs. Data are presented as mean ± SD. All experiments were run in triplicate, unless noted otherwise. Scale bars = 50 µm. ∗, p < .05 compared with the control media group. (E): Representative antibody array images showing the proteins presenting in the CM from LSCs and NHDF cells. Abbreviations: AQ5, aquaporin 5; BDNF, brain-derived neurotrophic factor; CM, conditioned media; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GRO, growth-regulated protein; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; LSCs, lung spheroid cells; POS, positive; NHDF, normal human dermal fibroblast cell.

Now the $64,000 question is this: “How do LSCs stack up against MSCs at treating lung damage?”

To address this question, Cheng and others used a mouse model of pulmonary fibrosis. To induce pulmonary fibrosis in mice, Severe Combined Immune Deficient (SCID) mice were given intratracheal washes of the anticancer drug bleomycin, which induces a condition in mice that, to some degree, resembles human pulmonary fibrosis. Different groups of mice with this lung damage were given either intravenous infusions of LSCs, MSCs or saline as a control.

The administered LSCs reduced the amount of cell death and scarring observed in the lungs. LSCs also increased the formation of new blood vessels and decreased the expression of pro-fibrotic genes. While the infusion of MSCs did improve the lung tissue in these mice, the LSCs were clearly superior.

Therapeutic benefits of human LSCs in mice with bleomycin-induced pulmonary fibrosis. (A): Schematic showing the design of the mouse studies. (B): Macroscopic views of explanted lungs 14 days after LSC or saline treatment. H&E staining (C) and Masson’s trichrome staining (D) were performed on the lungs. (E): Quantitation of fibrous thickening by Ashcroft score from the H&E staining images (n = 6–7 animals per group). (F): Quantitation of tissue infiltrates from the H&E staining images (n = 6–7 animals per group). Data are presented as mean ± SD. Scale bars = 100 µm. ∗, p < .05 compared with the sham group; #, p < .05 compared with the Bleo + saline group. Abbreviations: Bleo, bleomycin; H&E, hematoxylin and eosin; LSCs, lung spheroid cells.
Therapeutic benefits of human LSCs in mice with bleomycin-induced pulmonary fibrosis. (A): Schematic showing the design of the mouse studies. (B): Macroscopic views of explanted lungs 14 days after LSC or saline treatment. H&E staining (C) and Masson’s trichrome staining (D) were performed on the lungs. (E): Quantitation of fibrous thickening by Ashcroft score from the H&E staining images (n = 6–7 animals per group). (F): Quantitation of tissue infiltrates from the H&E staining images (n = 6–7 animals per group). Data are presented as mean ± SD. Scale bars = 100 µm. ∗, p < .05 compared with the sham group; #, p < .05 compared with the Bleo + saline group. Abbreviations: Bleo, bleomycin; H&E, hematoxylin and eosin; LSCs, lung spheroid cells.

Can LSCs provide the kind regenerative “OOMPH” damaged lungs need? The culture system that was developed by Cheng and others can produce large quantities of cells from even small biopsies. This makes the procedure suitable and efficacious for clinical situations in which a patient is receiving infusions of their own cells or someone else’s cells. An added advantage to this system in the absence of any need for cell sorting, which is expensive, and requires highly-trained technicians who operate large, expensive machines. Also, none of the mice treated in this study showed any signs of tumors, which underscores the clinical safety of LSCs.

Cultured MSCs might also provide an excellent model system to study lung pathologies. Making LSCs from patients with cystic fibrosis, inherited versions of emphysema, or other pulmonary diseases could provide an accessible and effective model system for drug testing and pathological studies.

This work was published in the journal Stem Cells and Translational Medicine.

New Way to Improve Stem Cell Production May Improve IPF Treatment


North Carolina State University researcher have tested a faster, cheaper way to harvest and grow lung stem cells that have been extracted from patients’ own bodies. That makes such cells a perfect match for lung patients, according to a small proof-of-concept trial.

Ke Cheng, an associate professor of regenerative medicine at NC State, and his team tested this method with, a view toward eventually treating people with idiopathic pulmonary fibrosis, or IPF, a disease that causes inflammation in lung tissue that over time becomes thick and stiff. This scarring of tissue negatively affects lung function over time.

“In current stem cell harvesting, just the process of sorting the stem cells can damage them, wasting not only the cells, but also time and money,” said Cheng. “We wanted to see if we could take healthy stem cells from an organ while they were still in a supportive environment, recreate and enhance that environment outside the body to encourage stem cell reproduction, then reintroduce those cells into a damaged organ to treat disease.”

Cheng and others placed healthy, human adult lung stem cells in a multicellular spheroid, a three-dimensional structure with stem cells in the middle surrounded by layers of support cells. Spheroids are typically used in the laboratory to culture cancer or embryonic cells.

They then used mice with IPF and injected cultured human stem cells into the animals. These injected stem cells produced decreases in inflammation and fibrosis, which Cheng said matched the condition of lungs in the study’s control group that did not have IPF.

Cheng hopes that stem cells isolated from biopsies in human patients can be used to grow and harvest additional cells. Such a procedure should be able to decrease the number of invasive procedures necessary for treatment.

“Picture the lung as a garden and the stem cells as seeds,” Cheng said. “In an IPF environment, with inflammation, the soil is bad, but the seeds are still there. We take the seeds out and give them a protected place to grow. Then when we put them back into the lung, they can grow into mature lung cells to replace the damaged lung tissues in IPF. They can also wake the other seeds up, telling them to help fight the inflammation and ‘improving’ the soil.”

The study was published in the journal STEM CELLS Translational Medicine.

Healing Damaged Lungs with Stem Cells


Emphysema, bronchitis, asthma and cystic fibrosis are all diseases of the airways and they are the second leading cause of death worldwide. Over 35 million Americans alone suffer from chronic respiratory disease.

Scientists from the Weizmann Institute have now proposed a new direction for treating these diseases that might lead to a new method for alleviating some of their suffering. The study’s findings, which were published in Nature Medicine, show that it might be possible to use fetal stem cells to repair damaged lung tissue.

Particular stem cells normally found in the lungs are highly similar to those in the bone marrow. Organ-specific stem cells tend to be concentrated in special compartments rather than being distributed throughout the tissue. This insight prompted Prof. Yair Reisner of the Weizmann Institute’s Immunology Department to suggest: “That understanding suggested to us that we might be able to apply our knowledge of techniques for transplanting bone marrow stem cells to repairing lung tissue.”

Bone marrow transplants are based on two main principles: the ability of stem cells to navigate through the blood to the appropriate compartment and the prior clearing out of the compartment to make room for the transplanted stem cells. Dr. Reisner and his group thought it might possible to apply these principles to introducing new stem cells into the lungs. However, before they could do this, they needed to find a source of lung stem cells suitable for transplanting.

Reisner and his co-workers used fetal lung tissue from mice and humans (20–22 weeks of gestation for humans, and embryonic day 15–16 or E15–E16 for mice). Cells from these stages have differentiated into lung progenitor cells and are fully capable of lung regeneration. Reisner and his colleagues conducted a series of experiments in which they cleared the lung’s stem cell compartments with a new method developed on their own laboratory, and then were injected these new lung progenitor cells into mouse models of lung damage. The fetal lung stem cells found their way through the blood to the lungs and settled into the proper compartment. By six weeks, these cells were well on their way to differentiating into normal lung tissue. In these mice, their damaged lungs healed, and their breathing improved significantly.

Next, Reisner intends to determine the correct dosage of drugs that are needed to prevent rejection of the transplanted cells, which will be needed following such procedures. “But our real vision, bolstered by this success,” says Reisner, “is to create a bank of lung tissue that will be a resource for embryonic lung stem cells.” This bank could mean that there is a ready source of cells for repairing the damage in those with severe respiratory disease.

Reisner’s work shows that fetal lung progenitors can repopulate lungs and heal them. If Reisner can find a way to generate early lung progenitors from pluripotent stem cells, then such cells can be used to heal damaged lungs.

One Type of Lung Cell Can Regenerate Another


A collaboration between the Perelman School of Medicine at the University of Pennsylvania and Duke University has found that lung tissue has a much great ability to regenerate than previously thought.

Lungs contain thousands of tiny clusters of sacs called alveoli. Gas-exchange between the air and our blood stream occurs across the thin lining of the alveoli, which are lined with extensive networks for diminutive blood vessels called capillaries. The cells that form the paper-thin lining of the alveoli are called type 1 cells. Within the alveoli are cells called type 2 cells, which secrete surfactant; a soapy substance that prevents the alveoli from collapsing upon themselves when we exhale. Some premature babies do not make enough surfactant and must be treated with surfactant to help them breathe.

Work in mice demonstrated that both type 1 and type 2 cells descend from a common embryonic precursor during lung development. When mice had bits of their lungs removed, labeling studies established that the newly re-established type 2 cells were made from type 1 cells and that some of the newly made type 1 cells were formed from type 2 cells. These results were confirmed by cell culture experiments that grew single type 1 or type 2 lung cells in culture; in both cases, the cultures gave rise to mixed cultures consisting of both type 1 and type 2 lung cells. These data demonstrate that type 1 lung cells can give rise to type 2 lung cells and visa versa.

Previously, the Duke University term had demonstrated that type 2 lung cells in mice not only produce surfactant, but also function as progenitors for other lung cells in adult mice. This shows that type 2 lung cells can definitely differentiate into type 1 lung cells. However, there was no evidence that type 1 lung cells could give rise to other types of lung cells.

In this present work, however, lung injury in mice stimulated the type 1 cells to divide and differentiate into type 2 cells over a period of three weeks while the lung regenerated. According to Jonathan Epstein from the University of Pennsylvania, It’s as if the lung cells can regenerate from one another as needed to repair missing tissue, suggesting that there is much more flexibility in the system than we have previously appreciated. These aren’t classic stem cells that we see regenerating the lung. They are mature lung cells that awaken in response to injury. We want to learn how the lung regenerates so that we can stimulate this process in situations where it is insufficient, such as in patients with COPD (chronic obstructive pulmonary disease).”

This is one of the first studies to demonstrate that mature cells that were thought to be completely at the end of their growth and differentiation capabilities can revert to an earlier state under the right conditions without the use of transcription factors, but by responding to damage.

These two research teams are also applying the approaches outlined in this publication to cells from other tissues, such as the intestine and skin, in order to study the mechanisms of cell maintenance and differentiation, and then relate these same mechanisms back to the heart. They also hope to apply these findings in clinical settings for patients who suffer from idiopathic pulmonary fibrosis, acute respiratory distress syndrome and other such conditions where the alveoli cannot supply sufficient amounts of oxygen to the blood.

Scientists Use Stem Cells to Grow Three-Dimensional Mini Lungs.


In research done in several laboratories, lung tissue was derived from flat cell culture systems or by growing cells on scaffolds made from donated organs.

Now in a new study published in the online journal eLife, a multi-institution team has defined a culture system for generating self-organizing human lung organoids, which are three-dimensional structures that mimic the structure and complexity of human lungs.

“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,” said study senior author Jason R. Spence, Ph.D., an assistant professor of internal medicine and cell and developmental biology at the University of Michigan Medical School.

Spence and his colleagues successfully grew structures that resembled both the large airways or bronchi and small lung sacs, known as alveoli.

These mini lung structures were developed in a cell culture system. Therefore, they lack several components of the human lung, including blood vessels, which are a critical component of gas exchange during breathing.

Despite that, these cultured organoids can serve as a unique research model system for researchers as they grind out basic science ideas that are turned into clinical innovations. These three-dimensional mini-lungs should be an excellent complement to research in liver laboratory animals.

Traditionally, the behavior of cells has been investigated in the laboratory in two-dimensional culture systems where cells are grown in thin layers on cell-culture dishes. Most cells in the body, however, exist in a three-dimensional environment as part of complex tissues and organs. Tissue engineered have been trying to re-create these environments in the laboratory by successfully generating small version of particular organs known as organoids, which serve as models of the stomach, brain, liver and human intestine. The advantage of growing three-dimensional structures of lung tissue, according to Dr. Spence, is that the organization of organoids bears greater similarity to the human lung.

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 cell signaling pathways that control the formation of organs.

First, stem cells were induced 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. Second, the group activated two important development pathways (FGF and WNT signaling ) that are stimulate endoderm to form three-dimensional tissue. By inhibiting two other key development pathways at the same time (BMP and TGFβ signaling), the endoderm became tissue that resembles the early lung found in embryos.

In the laboratory, this early culture-derived lung-like tissue spontaneously formed three-dimensional spherical structures as it developed. Afterwards, they had to expand these structures and develop them into lung tissue. In order to do this, Spence and his colleagues and collaborators exposed the cells to additional proteins involved in lung development (FGF and Hedgehog).

After all this manipulation, the resulting lung organoids survived in the laboratory for over 100 days.

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

While this type of experiment is remarkable, this is only the beginning of lung tissue engineering.  These mini-lungs  will hopefully serve and new model systems for drug testing and researching genetic diseases that affect the lungs, such as cystic fibrosis, sarcoidosis, or inherited forms of emphysema.  It will be a while before scientists can make replacement lungs for human patients, but these experiments by Spence and others are a remarkable start.

Rare Stem Cell Heals Damaged Lungs; Notch Signaling May Hold the Key to Lung Fibrosis


Patients who survive an acute lung injury are able to recover their lung function, which suggests that adult lungs regenerate to a certain extent. Depending on the cause and severity of the injury, multiple progenitor cells, including alveolar type II cells and distal airway stem cells, have been shown to drive lung tissue regeneration in mice. Now, Andrew Vaughan and others have described another cell type in the lungs involved in the repair process in mice when mouse lungs are damaged from influenza virus infection or inhalation of the anticancer drug bleomycin.  This cell type is called the rare lineage-negative epithelial progenitor (LNEP).

LNEP cells are quiescently present within normal distal mouse lung and do not express mature lineage markers (for example, a protein called club cell 10 or CC10 or surfactant protein C, otherwise known as SPC).  However, Vaughan and others demonstrate that LNEPs are activated to proliferate and migrate to damaged sites and mediate lung remodeling following major injury.

Vaughan and others used lineage tracing approaches and cell transplantation strategies and showed that LNEP cells, but not mature epithelial lineage cells, are multipotent in their ability to give rise to both club cells and alveolar cells.  Interestingly, activation of the Notch signaling pathway in LNEP cells initially activated them, but persistent Notch activation inhibited subsequent alveolar differentiation, resulting in failed tissue regeneration (characterized by the formation of abnormal honeycomb cysts in the mouse lung).  Thus Notch signaling is only required at the beginning of their activation, and then must be down-regulated if the LNEP cells are to reconstruct normal lung tissue.  Interestingly, scarred over or fibrotic lungs from patients with idiopathic pulmonary fibrosis or a disease called scleroderma show evidence of hyperactive Notch signaling and their lungs also contain very similar-looking honeycomb cysts.  This strongly suggests that dynamic Notch signaling also regulates the function and differentiation of LNEP-analogous human lung progenitor cells.  Thus designing treatments that properly regulate Notch signaling and, consequently, LNEP activity may potentially halt the development of lung fibrosis in humans.

Lung Stem Cells Heal Lungs and Point to Possible New Treatments


Frank McKeon, Ph.D., and Wa Xian, Ph.D. from Jackson Laboratory and their colleagues have identified the a certain lung stem cell, and the role it plays in regenerating lungs.

This work, which appeared in the Nov. 12 issue of the journal Nature, provides some much-needed clarification of the nuts and bolts of lung regeneration and provides a way forward for possible therapeutic strategies that harness these lung stem cells.

“The idea that the lung can regenerate has been slow to take hold in the biomedical research community,” McKeon says, “in part because of the steady decline that is seen in patients with severe lung diseases like chronic obstructive pulmonary disease (known as COPD) and pulmonary fibrosis.”

McKeon noted that there is ample evidence of a robust system for lung regeneration. “Some survivors of acute respiratory distress syndrome, or ARDS, for example, are able to recover near-normal lung function following significant destruction of lung tissue.”

This is a capacity that humans share with mice. Mice infected with the H1N1 influenza virus show progressive inflammation in the lung followed by the death and loss of important lung cell types. However, over the course of several weeks, the lungs of these mice recover and show no signs of previous lung injury.

Because of the presence of such robust lung regeneration in mice, these organisms provide a fine model system to study lung regeneration.

McKeon and his colleagues had previously identified a type of adult lung stem cell known as p63+/Krt5+ in the distal airways. When grown in culture, these p63+/Krt5+ lung stem cells neatly formed alveolar-like structures that were similar to those found within the lung. Alveoli are the tiny, specialized air sacs that form at the ends of the smallest airways, where gas exchange occurs in the lung. Following infection with H1N1, these same stem cells migrated to sites of inflammation in the lung and clustered together to form pod-like structures that resemble alveoli, both visually and molecularly.

McKeon and his colleagues reported that when the lung is damaged by H1N1 infraction, p63+/Krt5+ lung stem cells proliferate and contribute to the development of new alveoli near sites of lung inflammation.

To determine if these cells are required for lung regeneration, McKeon and his coworkers developed a novel system that utilizes genetic tools to selectively remove these cells from the mouse lung. Mice that lack p63+/Krt5+ lung stem cells cannot recover normally from H1N1 infection, and instead exhibit scarring of the lung and impaired oxygen exchange. This demonstrates the key role p63+/Krt5+ lung stem cells play in regenerating lung tissue.

To carry this work one step further, McKeon and his team isolated and subsequently transplanted p63+/Krt5+ lung stem cells into a damaged lung. The transplanted p63+/Krt5+ cells readily contribute to the formation of new alveoli, which nicely illustrates the capacity of these cells to regenerate damaged lung tissue.

In the U.S. about 200,000 people have Acute Respiratory Distress Syndrome, a disease with a death rate of 40 percent, and there are 12 million patients with COPD. “These patients have few therapeutic options today,” Xian says. “We hope that our research could lead to new ways to help them.”

MSCs for Tissue Engineered Tracheas and Enhanced Fracture Healing


For all my readers who have ever broken a bone, this one’s for you.

Setting a broken bone properly can lead to the healing of a broken bone, but large fractures that generate gaps in bones are very hard to heal. Stem cell therapy in combination with small protein molecules called cytokines has the potential to improve bone repair, since cytokines summon resident stem cells to migrate and home to the injured site. Having said that, the engraftment, participation and recruitment of other cells within the regenerating tissue are equally important.

To stimulate stem cell-mediated healing, University College London scientists over-expressed the SDF-1 protein in mesenchymal stem cells. Since SDF-1 is a stem cell-recruitment protein, it seems reasonable to suspect that these engineered cells would effectively increase the migration of native cells to the site of fracture and enhance bone repair.

Once they made SDF-1-expressing mesenchymal stem cells, Chih-Yuan Ho and colleagues showed that these cells increased the migration of non-transfected cells in a cell culture system.

Once these SDF-1-expressing mesenchymal stem cells were implanted into rats with large bone defects, bone marrow mesenchymal stem cells that over-expressed SDF-1 showed significantly more new bone formation within the gap and less bone mineral loss at the areas next to the defect site during the early bone healing stage.

Thus, SDF-1 plays an important role in accelerating fracture repair and contributing to bone repair, at least in this rat model. SDF-1 does this by recruiting more host stem cells to the defect site and encouraging their differentiation into bone cells, which go on to produce good-quality bone.  This paper appeared the the journal Tissue Engineering, Part A.

In a second paper that appeared in the Annals of Biomedical Engineering, mesenchymal stem cells were used to tissue engineer tracheae. In this case a biocompatible scaffold was seeded various with various cells and this strategy could be a solution for tracheal reconstruction.

Yoo Seob Shin and colleagues seeded mesenchymal stem cells (MSCs) on a scaffold made from pig cartilage powder (PCP). The PCP was made with minced and decellularized pig joint cartilage and was molded into a 5 × 12 mm (height × diameter) scaffold. Mesenchymal stem cells from the bone marrow of young rabbits were grown in culture and then cultured with the PCP scaffold. After 7 weeks in culture, these tracheal implants were transplanted on a 5 × 10 mm tracheal defect in six rabbits, which were evaluated 6 and 10 weeks after the operation.

None of the six rabbits showed any sign of respiratory distress, and endoscopic examination of these tissue engineered tracheae showed that the a normal-looking respiratory epithelium completely covered the regenerated trachea. These trachea also displayed no signs of collapse or blockage.

The tissue engineered tracheae were also scanned and modeled on a computer model (luminal contour). The reconstructed areas of the trachea were the right width and dimensions compared to normal adjacent trachea and were not narrow.

Detailed microscopic tissue examinations of the tissue engineered tracheae showed that the new cartilage was successfully produced by the seeded mesenchymal stem cells and there was only a minimal degree of inflammation or granulation tissue that forms on the surfaces of wounds during the healing process. This shows that the implants did not trigger a massive inflammatory response that damaged resident or implanted tissue.

The outer surfaces of tracheal cells are decorated with tiny beating hairs called cilia that constantly beat to clear particles from the respiratory system. There are also cells that secrete mucus, which acts like fly paper for invading pollutants, particles or microorganisms. in the tissue engineered tracheae, ciliary beating frequency of the regenerated epithelium was not significantly different from the normal adjacent mucosa.

Thus, mesenchymal stem cells from bone marrow seeded on a PCP scaffold successfully restored not only the shape but also the function of the trachea without any signs of graft rejection.

Bones and trachea – mesenchymal stem cells pack a powerful healing punch!!

FDA Approves Pneumostem Clinical Trial for Bronchopulmonary Dysplasia


MEDIPOST America Inc. has announced that the US Food and Drug Administration (USFDA) has approved their product Pneumostem for a Phase 1/2 clinical trial. This Phase 1/2 trial will assess the safety and efficacy of Pneumostem on prematurely born infants who are at high-risk of developing Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious lung condition that affects infants. BPD usually affects premature infants who need oxygen given through nasal prongs, a mask, or a breathing tube in order to properly breathe.

Most infants who develop BPD are born more than 10 weeks before their due dates and weigh less than 2 pounds (about 1,000 grams) at birth, and have breathing problems. Respiratory infections that hit before or shortly after birth also can contribute to BPD.

Some infants who suffer from BPD may need long-term breathing support from breathing (NCPAP) machines or ventilators. BPD is the leading cause of mortality and severe complications in premature infants. Currently there is no approved therapies or drugs exist for BPD. This pneumostem trial is expected to draw global attention in the field of neonatal medicine, since it would provide a potential treatment for BPD where none presently exists.

Pneumostem is an off-the-shelf product made from human Umbilical Cord Blood-derived Mesenchymal Stem Cells (hUCB-MSCs). hUCB-MSCs show a terrific ability to grow in the laboratory and can also differentiate into multiple types of cells or tissues. They are immune-privileged and thus if they are used in patients other than from whom they are isolated, they do not cause adverse immune reactions. hUCB-MSCs harvested from cord blood show the lowest levels of immunogenicity compared to those by other types of adult stem cells. Thus, instead of provoking immunogenicity, they rather modulate the adverse immune reactions within the host, which makes hUCB-MSCs an ideal candidates for mass-producible stem cell drug for allogeneic use. These cells seem to facilitate regeneration of lung tissue and suppress the inflammatory responses in the lungs of premature infants.

Pneumostem has received Orphan Drug designation in Korea by the Ministry of Food and Drug Safety (MFDS) and the Korean Phase 2 study is 80% complete. The US FDA also granted Orphan Drug designation for Pneumostem demonstrating its medical value and commercial potential.

Presently, MEDIPOST America is rapidly moving begin this Pneumostem trial in the U.S. At the same time, Medipost will continue its licensing and technology transfer negotiations with multinational pharmaceutical companies.

The approval of this Pneumostem clinical trial by the US FDA, whose regulation of medicinal products is very strict (including stem cell products), might boost clinical trial approvals in other European and Asian countries.

Clinical development of Pneumostem was partly supported by Translational Stem Cell & Regenerative Medicine Consortium grant as a part of Public Health and Medicinal Technology R&D Project funded by the Korea Ministry of Health & Welfare and the Korea Health Industry Development Institute.

Induced Pluripotent Stem Cells Make Lungs


Since my father died of disseminated lung cancer (squamous cell carcinoma), this report has particular meaning to me.

When a person dies, their lungs can be harvested and stripped of their cells. This leaves a so-called “lung scaffold” that can then be used to build new lungs by means of tissue engineering techniques. Lung scaffolds consist of a protein called collagen, and sugar-rich proteins called “proteoglycans” (say that fast five times) and a rubber band-like protein called elastin. Depending on how the lung scaffolds are made more or less of these components can remain in the lung scaffold (see TH Peterson, and others, Cells Tissues Organs. Feb 2012; 195(3): 222–231). The important thing is that the cells are gone and this greatly reduces the tendency for the lung scaffold to be rejected by someone else’s immune system.

Once a lung scaffold is generated from a whole lung, cells can be used to reconstitute the lung. The key is to use the right cell type or mix of cell types and to induce them to form mature lung tissue.

The laboratory of Harald Ott at Harvard University Medical School used a technique called “perfusion decellularization” to make lung scaffolds from the lungs of cadavers. Then he and his co-workers used lung progenitor cells that were derived from induced pluripotent stem cells (iPSCs). This study was published in The Annals of Thoracic Surgery, and it examined the ability of iPSCs to regenerate a functional pulmonary organ

Whole lungs from rat and human cadavers were stripped of their living material by means of constant-pressure perfusion with a strong detergent called sodium dodecyl sulfate (SDS; 0.1% if anyone is interested). Ott and his crew then sectioned some of the resulting lung scaffolds and left others intact, and then applied human iPSCs that had been differentiated into developing lung tissue.

Lung tissue develops from the front part of the developing gut. This tissue is called “endoderm,” since it is in the very innermost layer of the embryo.

Lung Development

Therefore, the iPSCs were differentiated into endoderm with a cocktail of growth factors (FGF, Wnt, Retinoic acid), and then further differentiated in the anterior endoderm (foregut; treated cells with Activin-A, followed by transforming growth factor-β inhibition), and then even further differentiated into anterior, ventral endoderm, which is the precise tissue from which lungs form. In order to be sure that this tissue is lung tissue, they must express a gene called NK2 homeobox 1 (Nkx2.1). If these cells express this gene, then they are certainly lung cells.

Ott and his group showed that their differentiate iPSCs strongly expressed Nkx2.1, and then seeded them on slices and whole lung scaffolds. Then Otts’s group maintained these tissues in a culture system that was meant to mimic physiological conditions.

Those cells cultured on decellularized lung slices divided robustly and committed to the lung lineage after 5 days. Within whole-lung scaffolds and under the physiological mimicking culture, cells upgraded their expression of Nkx2.1. When the culture-grown rat lungs were transplanted into rats, they were perfused and ventilated by host vasculature and airways.

Thus these decellularized lung scaffolds supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Furthermore, whole-organ scaffolds and a culture system that mimics physiological conditions, allows scientists to enable seeding a combination of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Transplantation of these laboratory-grown lungs seem to further maturation of these grafted lung tissues.

Reversing Lung Diseases By Directing Stem Cell Differentiation


Lung diseases can scar the respiratory tissues necessary for oxygen exchange. Without proper oxygen exchange, our cells lack the means to make the energy they so desperately need, and they begin to shut down or even die. Lung diseases such as asthma, emphysema, chronic obstructive pulmonary disease and others can permanently diminish lung capacity, life expectancy and activity levels.

Fortunately, a preclinical study in laboratory animals has suggested a new strategy for treating lung diseases. Carla Kim and Joo-Hyeon Lee of the Stem Cell Research Program at Boston Children’s have described a new lung-specific pathway that is activated by lung injury and directs a resident stem cell population in the lung to proliferate and differentiate into lung-specific cell types.

When Kim and Lee enhanced this pathway in mice, they observed increase production of the cells that line the alveolar sacs where gas exchange occurs. Alveolar cells are irreversibly damaged in emphysema and pulmonary fibrosis.

Inhibition of this same pathway increased stem cell-mediated production of airway epithelial cells, which line the passages that conduct air to the alveolar sacs and are damaged in asthma and bronchiolitis obliterans.

For their experiments, Kim and Lee used a novel culture system called a 3D culture system that mimics the milieu of the lung. This culture system showed that a single bronchioalveolar stem cell could differentiate into both alveolar and bronchiolar epithelial cells. By adding a protein called TSP-1 (thrombospondin-1), the stem cells differentiated into alveolar cells.

Next, Kim and Lee utilized a mouse model of pulmonary fibrosis. However, when they cultured the small endothelial cells that line the many small blood vessels in the lung, which naturally produce TSP-1, and directly injected the culture fluid of these cells into the mice, the noticed these injections reverse the lung damage.

When they used lung endothelial cells that do not produce TSP-1 in 3D cultures, lung-specific stem cells produce more airway cells. in mice that were engineered to not express TSP-1, airway repair was enhanced after lung injury.

Lung Stem Cell Repair of Lung Damage

Lee explained his results in this way: “When the lung cells are injured, there seems to be a cross talk between the damaged cells, the lung endothelial cells and the stem cells.”

Kim added: “We think that lung endothelial cells produce a lot of repair factors besides TSP-1. We want to find all these molecules, which could provide additional therapeutic targets.”

Even though this work is preclinical in nature, it represents a remarkable way to address the lung damage that debilitates so many people. Hopefully this work is easily translatable to human patients and clinical trials will be in the future. Before that, more confirmation of the role of TSP-1 is required.

Stem Cells Heal Damaged Cells by Transferring Mitochondria


An Indian team from Delhi, India has identified a protein that increases the transfer of mitochondria from mesenchymal stem cells to lung cells, thus augmenting the healing of lung cells.

Stem cells like mesenchymal stem cells from bone marrow, fat, tendons, liver, skeletal muscle, and so on secrete a host of healing molecules, but they also form bridges to other cells and export their own mitochondria to heal damaged cells. Mitochondria are the structures inside cells that make energy. Damaged cells can have serious energy deficiencies and mitochondrial transfer ameliorates such problems (see Cárdenes N et al, Respiration. 2013;85(4):267-78).

This present work from the laboratory of Anurag Agrawal, who is housed in the Centre of Excellence in Asthma & Lung Disease, at the CSIR‐Institute of Genomics and Integrative Biology in Delhi, India has identified a protein called Miro1 that regulates the transfer of mitochondria to recipient cells.

Mitochondrial transfer has so many distinct benefits that stem cell scientists hope to engineer stem cells to transfer more of their mitochondria to damaged cells, and Miro1 might be a target for such stem cell engineering experiments.

Mitochondrial transfer between stem cells and other cells occurs by means of tunneling nanotubes, which are thread-like structures formed from the plasma membranes of cells that form bridges between different cell types. Under stressful conditions, the number of these nanotubes increases.

In the present study. stem cells engineered to express more Miro1 protein transferred mitochondria more efficiently than control stem cells. When used in mice with damaged lungs and airways, these Miro1-overexpressing cells were therapeutically more effective than control cells.

This study presents the first mechanistic insight into how Mesenchymal Stem Cells (MSC) act as mitochondrial donors during attenuation of lung inflammation and injury. Mitochondrial donation is an essential part of the MSC therapeutic effect in these models and is positively regulated by Miro1 / Rhot1 mitochondrial transport proteins.
This study presents the first mechanistic insight into how Mesenchymal Stem Cells (MSC) act as mitochondrial donors during attenuation of lung inflammation and injury. Mitochondrial donation is an essential part of the MSC therapeutic effect in these models and is positively regulated by Miro1 / Rhot1 mitochondrial transport proteins.

The hope is to use Miro1 manipulations to make better stem cell therapies for human diseases.

To summarize this work:

1. MSCs donate mitochondria to stressed epithelial cells (EC) that have malfunctioning mitochondrial.  Cytoplasmic nanotubular bridges form between the cells and Miro‐1 mediated mitochondrial transfer occurs unidirectionally from MSCs to ECs.

2. Other mesenchymal cells like smooth muscle cells and fibroblasts express Miro1 and can also donate mitochondria to ECs, but with low efficiency. ECs have very low levels of Miro1 and, as a rule, do not donate mitochondria.

3. Enhanced expression of Miro1 in mesenchymal cells increases their mitochondrial donor efficiency.  Conversely, cells lacking Miro1 do not show MSC mediated mitochondrial donation.

4. Miro1‐overexpressing MSCs have enhanced therapeutic effects in three different models of allergic lung inflammation and rat poison-induced lung injury.  Conversely, Miro1‐depleted MSCs lose much of their therapeutic effect.  Miro1 overexpression in MSCs may lead to more effective stem cell therapy.

Human Stem Cells Converted into Functional Lung Cells


Scientists from the Columbia University Medical Center have succeeded in transforming human stem cells into functional lung and airway cells. This finding has significant potential for modeling lung disease, screening lung-specific drugs, and, hopefully, generating lung tissue for transplantation.

Study leader, Hans-Willem Snoeck, professor of medicine and affiliated with the Columbia Center for Translational Immunology and the Columbia Stem Cell Initiative, said, “Researchers have had relative success in turning human stem cells into heart cells, pancreatic beta cells, intestinal cells, liver cells, and nerve cells, raising all sorts of possibilities for regenerative medicine. Now, we are finally able to make lung and airway cells. This is important because lung transplants have a particularly poor prognosis. Although any clinical application is still many years away, we can begin thinking about making autologous lung transplants – that is, transplants that use a patient’s own skin cells to generate functional lung tissue.”

The research builds on Snoeck’s earlier discoveries in 2011 that a set of chemical factors could induce the differentiation of embryonic or induced pluripotent stem cells into “anterior foregut endoderm,” which is the embryo in the tissue from which the lungs form (Green MD, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011 Mar;29(3):267-72).

Human Embryological Development - one month

In his new study, Snoeck and his colleagues found new factors that can transform anterior foregut endoderm cells into lung and airway cells. In particular, Snoeck and his co-workers were able to establish the presence of “type 2 alveolar epithelial cells,” which secrete the lung surfactant that maintains the lung alveoli (those tiny sacs in the lung where all the oxygen exchange takes place).

lung alveolus

With these techniques, lung researchers hope to study diseases like idiopathic pulmonary fibrosis (IPF), in which type 2 epithelial cells seem to divide and produce scarring in the lungs.

“No one knows what causes the disease, and there’s no way to treat it,” said Snoeck. “Using this technology, researchers will finally be able to create laboratory models of IPF, study the disease at the molecular level, and screen drugs for possible treatments or cures. In the longer term, we hope to use this technology to make an autologous lung graft. This would entail taking a lung from a donor, removing all the lung cells, leaving only the lung scaffold; and seeding the scaffold with new lung cells derived from the patient. In this way, rejection problems could be avoided.”

Snoeck is investigating this approach in collaboration with researchers in the Columbia University Department of Biomedical Engineering.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Bioengineered Trachea Implanted into a Child


Hannah Genevieve Warren was born in 2010 in Seoul, South Korea with tracheal agenesis, which is to say that she was born without a trachea. Hannah had a tube inserted through her esophagus to her lungs that allowed her to breathe. Children with tracheal agenesis usually die in early childhood, 100% of the time. No child with this condition has ever lived past six years of life. Hannah spent the first two years of her life at the Seoul National Hospital before she was transported to Illinois for an unusual surgery.

While at the Children’s Hospital of Illinois, on April 9, 2013, Hannah had a bioengineered trachea transplanted into her body. This trachea was the result of a remarkable feat of technology called the InBreath tracheal scaffold and bioreactor system that was designed and manufactured by Harvard Bioscience, Inc. Harvard Bioscience, or HBIO, is a global developer, manufacturer and marketer of a broad range of specialized products, primarily apparatus and scientific instruments, used to advance life science research and regenerative medicine.

InBreath tracheal scaffold
InBreath tracheal scaffold

Hannah’s tracheal transplant was the first regenerated trachea transplant surgery that used a biomaterial scaffold that manufactured by the Harvard Apparatus Regenerative Technology (HART) Inc., a wholly owned subsidiary of Harvard Bioscience. HART ensured that the scaffold and bioreactor were custom-made to Hannah’s dimensions. Then the scaffold was seeded with bone marrow cells taken from Hannah’s bone marrow, and the cells were incubated in the bioreactor for two days prior to implantation. Because Hannah’s own cells were used, her body accepted the transplant without the need for immunosuppressive (anti-rejection) drugs.

InBreath Bioreactor
InBreath Bioreactor

The surgeons who participated in this landmark transplant were led by Dr. Paolo Macchiarini of Karolinska University Hospital and Karolinska Institutet in Huddinge, Stockholm and Drs. Mark J. Holterman and Richard Pearl both of Children’s Hospital of Illinois. This surgery was approved by the FDA under an Investigational New Drug (IND) application submitted by Dr. Holterman.

Dr. Mark Holterman, Professor of Surgery and Pediatrics at University of Illinois College of Medicine at Peoria, commented: “The success of this pediatric tracheal implantation would have been impossible without the Harvard Bioscience contribution. Their team of engineers applied their talent and experience to solve the difficult technical challenge of applying regenerative medicine principles in a small child.”

David Green, President of Harvard Bioscience, said: “We would like to congratulate Dr. Macchiarini, Dr. Holterman, Dr. Pearl and their colleagues for accomplishing the world’s first transplant of a regenerated trachea in a child using a synthetic scaffold and giving Hannah a chance at a normal life. We also wish Hannah a full recovery and extend our best wishes to her family.”

Hannah’s surgery is the seventh successful implant of a regenerated trachea in a human using HART technology. Prior successes included the first ever successful regenerated trachea transplant in 2008, the first successful regenerated trachea transplant using a synthetic scaffold in 2011, and the commencement of the first clinical trial of regenerated tracheas in 2012. HART has plans to commence discussions with the FDA and EU regulatory authorities in the near future regarding the clinical pathway necessary to bring this new therapeutic approach to a wider range of patients who are in need of a trachea transplant.