Patient-Specific Heart Cells Made from Amniotic Fluid Cells Before a Baby is Born

The dream of cardiologists is to have stockpiles of cardiac progenitor cells that could be transplanted into a sick heart and regenerate it. Even more remarkable would be a source of heart cells for newborn babies with congenital heart problems. What about making these cells before they are born? Science fiction?

Probably not. Dr. Shaun M. Kunisaki from Mott Children’s Hospital and the University of Michigan School of Medicine and his colleagues made heart progenitor cells from Amniotic Fluid Cells. These cells were acquired from routine amniocentesis procedures, with proper institutional review board approval.

These amniotic fluid specimens (8–10 ml), which were taken from babies at 20 weeks gestation, were expanded in culture and then reprogrammed toward pluripotency using nonintegrating Sendai virus (SeV) vectors that expressed the four commonly-used reprogramming genes; OCT4, SOX2, cMYC, and KLF4. The resulted induced pluripotent stem cell (iPSC) lines were then exposed to cardiogenic differentiation conditions in order to generate spontaneously beating amniotic fluid-derived cardiomyocytes (AF-CMs). AF-CMs were formed with high efficiency.

After 6 weeks, Kunisaki and his team subjected their AF-CMs to a battery of quantitative gene expression experiments. They discovered that their AF-CMs expressed high levels of heart-specific genes (including MYH6, MYL7, TNNT2, TTN, and HCN4). However, Kunisaki and others also found that their AF-CMs consisted of a mixed population of differentiated atrial, ventricular, and nodal cells, as demonstrated by various genes expression profiles.

All AF-CMs were chromosomally normal and had no remnants of the SeV transgenes. Functional characterization of these AF-CMs showed a higher spontaneous beat frequency in comparison with heart cells made from dermal fibroblasts. The AF-CMs also showed normal calcium currents and appropriately responded to neurotransmitters that usually speed up the heart, like norepinephrine.

Collectively, these data suggest that human amniotic fluid-derived cells can be used to produce highly scalable sources of functional, transgene-free, autologous heart cells before child is born. Such an approach may be ideally suited for patients with prenatally diagnosed cardiac anomalies.

Key Molecules Tha Control Stem Cell Fate Identified

Adult stem cells, such as mesenchymal stem cells and blood-vessel-associated pericytes represent patient-specific stem cells that are excellent candidates for regenerative medicine. To that end being able to control the differentiation of these stem cells with drugs or small molecules is extremely desirable for eliciting targeted tissue and organ regeneration.

However, identifying these stem-cell-inducing molecules is time-consuming, expensive, and fraught with dead ends. Is there an easier way to control the behavior of stem cells in culture or in your own body?

Research from the City University of New York (CUNY) suggests that the answer to this question might be “yes.” According to Rein Ulijn from CUNY, “Simple small metabolites present in the body already can dictate cell behavior.”

In collaboration with Matthew Dalby from the University of Glasgow, Ulijn and his colleagues discovered that when they grew stem cells on a gel-like medium, the stiffness of which could be easily adjusted, they found molecules that could direct the differentiation of cultured stem cells. As an added bonus, they could direct the differentiation of cultured stem cells much more cheaply.

Ulijn and Dalby began their collaboration in 2011 after other laboratories had demonstrated that the stiffness of the medium could affect the differentiation of stem cells. “On a stiff gel you might get bone-like differentiation,” Ulijn explained. “On a softer gel differentiation into neurons is more likely.” They wanted to use such a system to identify small molecules that can control stem cell differentiation in culture. Such a finding could also “aid the discovery of natural metabolite-based drugs,” added Ulijn added. Such natural-based drugs could be used to, for example, reinforce bones in osteoporosis.

Dalby was interested in the role metabolites played in this stem cell differentiation. Unfortunately, these metabolites are present in fleetingly low concentrations. To complicate the picture, the different formulations of stiffer and floppier materials can mask subtle changes in metabolite concentration. Ulijn found a way around this problem by turning to the two-component peptide gels made by Biogelx (full disclosure: Ulijn serves as the chief scientific officer for Biogelx). Fine-tuning the concentration of the two different gel components changes the rigidity of the gel without changing any other components of the gel that might mask metabolite variation.

The researchers therefore studied concentration changes of hundreds of metabolites during stiffness-controlled stem cell differentiation of stem cells into bone or cartilage. Several metabolites that seemed to make a significant difference for stem cell differentiation were lysophosphatidic acid, which drove stem cells to form cartilage and cholesterol sulfate, which helped stem cells form bone. When Ulijn and his coworkers fed these metabolites to standard stem cell cultures, they differentiated into the desired cell type.

Helena Azevedo of Queen Mary University of London, said, “We will see, for sure, studies exploiting these metabolites for inducing controlled differentiation of stem cells.” She went on to called this study “highly innovative” and said that it might directly influence future stem cell differentiation experiments; particularly those that involve the formation of cartilage or bone.

The Founder Cell Identity Does Not Affect iPS Cell Differentiation to Hematopoietic Stem Cell Fate

Induced pluripotent stem cells (iPSCs) have many of the characteristics of embryonic stem cells, but are made from mature cells by means of a process called cell reprogramming. To reprogram cells, particular genes are delivered into mature cells, which are then cultured until they h:ave the growth properties of pluripotent cells. Further tests are required to demonstrate that the growing cells actually are iPSCs, but once they pass these tests, these cells can be grown in culture indefinitely and, ideally, differentiated into just about any cell type in our bodies (caveat: some iPSC lines can only differentiate into particular cell lineages). Theoretically, any cell type can be reprogrammed into iPSCs, but work from many laboratories has demonstrated that the identity of the founder cell influences the type of cell into which it can be reprogrammed.

Founder cells can be easily acquired from a donor and come in one of four types: fibroblasts (in skin), keratinocytes (also from skin), peripheral and umbilical cord blood, and dental pulp cells (from baby teeth). A variety of laboratories from around the world have made iPSC lines from a gaggle of different founder cells. Because of the significant influence of founder cells for iPSC characteristics, the use of iPSCs for regenerative medicine and other medical applications requires that the desired iPSC line should be selected based on the founder cell type and the characteristics of the iPSC line.

However, the founder cell identity is not the only factor that affects the characteristics of derived iPSC lines. The methods by which the founder cells are reprogrammed can also profoundly contribute to the differentiation efficiency of iPSC lines. According to Yoshinori Yoshida, Associate Professor at the Center for iPSC Research and Application (CiRA) at Kyoto University, the most commonly used methods of cell reprogramming utilize retroviruses, episomal/plasmids, and Sendai viruses to move genes into cells.

The cells found in blood represent a diverse group of cells that includes red blood cells that carry oxygen, platelets that heal wounds, and white blood cells that fight off infection. All the cells in blood are made by bone marrow-specific stem cells called “hematopoietic stem cells.” The production of clinical grade blood has remained a kind of “holy grail” for cellular reprogramming studies. Some scientists have argued that in order to make good-quality hematopoietic cells, the best founder cells are hematopoietic cells. Is this true? Yoshida and his colleagues examined a very large number of iPSC lines that were made from different founder cells and with differing reprogramming methods.  The results of these experiments were published in the journal Cell Stem Cell (doi:10.1016/j.stem.2016.06.019).

Remarkably, Yoshida and his crew discovered that neither of these factors has a significant effect. What did have a significant effect were the expression of certain genes and the position of particular DNA methylations. These two factors were better indicators of the efficiency at which an iPSC line could differentiate into the hematopoietic stem cells.

“We found the IGF2 (Insulin-like Growth Factor-2) gene marks the beginning of reprogramming to hematopoietic cells”, said Dr. Masatoshi Nishizawa, a hematologist who works in Yoshida’s lab and is the first author of this new study. Higher expression of the IGF2 gene is indicative of iPSCs initiating differentiation into hematopoietic cells. Even though IGF2 itself is not directly related to hematopoiesis, its uptake corresponded to an increase in the expression of those genes involved in directing differentiation into hematopoietic stem cells.

Although IGF2 marked the beginnings of differentiation to hematopoietic lineage, the completion of differentiation was marked by the methylation profiles of the iPS cell DNA. “DNA methylation has an effect on a cell staying pluripotent or differentiating,” explained Yoshida. Completion of the final stages of differentiation was highly correlated with less aberrant methylation during the reprogramming process. Blood founder cells showed a much lesser tendency to display aberrant DNA methylation patterns than did other iPSC lines made from other founder cells. This probably explains why past experiments seemed to indicate that the founder cell contributes to the effectiveness of differentiating iPS cells to the hematopoietic stem cell lineage.

These findings reveal molecular factors that can be used to evaluate the differentiation potential of different iPSC lines, which should, hopefully, expedite the progression of iPSCs to clinical use. Nishizawa expects this work to provide the basis for evaluating iPSC lines for the preparation of other cell types. “I think each cell type will have its own special patterns,” he said.

Breakthrough in scaling up life-changing stem cell production

Research teams at the University of Nottingham, Uppsala University and GE Healthcare in Sweden have discovered a new method that could solve the big problem of the large-scale stem cell production required to fully realize the potential of these remarkable cells for understanding and treating disease.

Human pluripotent stem cells are undifferentiated and possess the unique potential to differentiate into all the different cell types of the body. With applications in disease modeling, drug screening, regenerative medicine and tissue engineering, there is an enormous demand for these cells, which will only grow as clinical applications and the pharmaceutical industry increase the use of these cells.

However, large-scale production of stem cells is not currently feasible because available culture methods are either too expensive, or rely on materials that are not be safe for clinical use in humans, such as animal-based proteins.

In this new publication, which appeared on Wednesday July 13 2016 in Nature Communications, a collaborative team that consisted of researchers from The University of Nottingham’s Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, Uppsala University and GE Healthcare have identified an improved method for human stem cell culture that, at least in principle, provide a faster and cheaper way for grow stem cells for large-scale industrial production.

The project had its genesis at Uppsala University in Sweden, and the first author, Dr Sara Pijuan-Galitó, is now continuing her work as a Swedish Research Council Research Fellow at Nottingham. Sara said: “By using a protein derived from human blood called Inter-alpha inhibitor, we have grown human pluripotent stem cells in a minimal medium without the need for costly and time-consuming biological substrates. Inter-alpha inhibitor is found in human blood at high concentrations, and is currently a by-product of standard drug purification schemes.

“The protein can make stem cells attach on unmodified tissue culture plastic, and improve survival of the stem cells in harsh conditions. It is the first stem cell culture method that does not require a pre-treated biological substrate for attachment, and therefore, is more cost and time-efficient and paves the way for easier and cheaper large-scale production.”

Lead supervisor Dr Cecilia Annerén, who has a joint position at Uppsala University and at GE Healthcare in Uppsala, said: “As coating is a time-consuming step and adds cost to human stem cell culture, this new method has the potential to save time and money in large-scale and high-throughput cultures, and be highly valuable for both basic research and commercial applications.”

Co-author on the paper Dr Cathy Merry added: “We now intend to combine Inter-alpha inhibitor protein with our innovative hydrogel technology to improve on current methods to control cell differentiation and apply it to disease modelling. This will help research into many diseases but our focus is on understanding rare conditions like Multiple Osteochondroma (an inherited disease associated with painful lumps developing on bones) at the cellular level. Our aim is to replicate the three-dimensional environments that cells experience in the body so that our lab-bench biology is more accurate in modelling diseases.”

Dr Sara Pijuan-Galitó’s next task is to combine the Inter-alpha inhibitor with improved synthetic polymers in collaboration with other regenerative medicine pioneers at the University, Professor Morgan Alexander and Professor Chris Denning. This team plans to further improve current human stem cell culture methods. Their goal is to design an economical and safe method that can be easily translated to large-scale production and deliver the billions of cells necessary to start taking cellular therapeutics to individual patients.