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.

New US Phase IIa Trial and Phase III Trial in Kazakhstan Examine CardioCell’s itMSC Therapy to Treat Heart Attack Patients


The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.

CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.

Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.

“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”

The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).

“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”

Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”

CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.

Researchers Create Inner Ear Structures From Stem Cells


Indiana University scientists have used mouse embryonic stem cells to make key structures of the inner ear. This accomplishment provides new insights into the sensory organ’s developmental process and sets the stage for laboratory models of disease, drug discovery and potential treatments for hearing loss, and balance disorders.

Eri Hashino, professor of otolaryngology at the University of Indiana School of Medicine, and his co-workers, were able to use a three-dimensional cell culture method that directed the stem cells to form inner-ear sensory epithelia that contained hair cells and supporting cells and neurons that detect sound, head movements and gravity.

In the past, other attempts to grow inner-ear hair cells in standard culture systems have not succeeded. Apparently the cues required to form inner-ear hair bundles, which are essential for detecting auditory or vestibular signals, are absent in cell-culture dishes.

Inner ear hair cells
Inner ear hair cells

To conquer this barrier, Hashino and his team changed their culture system. The suspended the cells as aggregates in a specialized culture medium and this mimicked conditions normally found in the body as the inner ear develops.

Another strategy that paid off was to precisely time the application of several small molecules that coaxed the stem cells to differentiate from one stage to the next into precursors for the inner ear.

a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j).  Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.
a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j). Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.

Even though the added growth factors made a big difference to the success of this experiment, it was the three-dimensional suspension culture system that provided many important mechanical cues. The tension caused by the pull of the cells on each other played a very important role in directing the differentiation of the cells to become inner-ear precursors.

Karl A Koehler, first author of this paper and a graduate student in the medical neuroscience program at IU School of Medicine said: “The three-dimensional culture allows the cells to self-organize into complex tissues using mechanical cues that are found during embryonic development.”

Hashino added that they were “surprised to see that once stem cells are placed in 3-D culture, these cells behave as if they knew not only how to self-organize into a pattern remarkably similar to the native inner ear.” Hashino continued: “Our initial goal was to make inner-ear precursors in culture, but when we did testing we found thousands of hair cells in a culture dish.”

Electrophysiological testing of these stem cell-derived hair cells showed that they were, in fact, functional, and were similar to those that sense gravity and motion. Moreover, neurons like those that normally link the inner-ear cells to the brain had also developed in their cell culture system, and were connected to the hair cells.

Hashino thinks that additional research is needed to determine how to derived inner-ear cells involved in auditory sensation might be made from stem cells, and how such techniques might be adapted to make human inner ear cells.