Making Cardiovascular Progenitor Cells from Induced Pluripotent Stem Cells

In fetal heart, stem cells known as cardiovascular progenitor cell (CPC) differentiates into smooth muscle cells for blood vessels, blood vessel wall cells, and heart muscle cells. Making CPCs from stem cells has proven to be rather difficult because CPCs do not express any known surface molecules that distinguishes them from other cell types. Therefore, if you want to differentiate pluripotent stem cells into CPCs, determining that you have made CPCs is very difficult.

This problem has been addressed by an international research team led by a team from Stuttgart, Germany who have discovered cell surface molecules that allow the identification and isolation of CPCs. With this knowledge, it will be possible to derive CPCs from induced pluripotent stem cells, which can be implanted into damaged hearts, differentiate into heart-specific cell types and integrate into the heart.

Heart attacks are the most frequent cause of death in the developed world. The cause of a heart attack is usually a clogged coronary vessel, which prevents sufficient blood flow through the heart and kills off heart tissue as a result of ischemia. There are some 17 million people who die from cardiovascular disease each.

Heart muscle cells (cardiomyocytes) do not have the ability to regenerate sufficiently after a heart attack. A heart attack causes a huge loss of cells and further impairs blood supply through the heart. This causes the heart to deteriorate further. To fix the heart, new heart muscle cells are required to replace to dead ones.

This now seems to be a distinct possibility. A research team led by Dr. Katja Schenke-Layland from the Frauhofer Institute for Interfacial engineering and Biotechnology IGD in Stuttgart, in collaboration with Dr. Ali Nasar from the University of California and Dr. Robb MacLean from the University of Washington in Seattle have used cultured CPCs to make heart muscle cells.

To identify CPCs, two proteins of the surfaces of CPCs were identified; a receptor called Flt1 and another called Flt4. By exploiting these two surface proteins, scientists can identify and isolate CPCs from a culture of differentiating pluripotent stem cells. To exploit this new finding, these groups, made induced pluripotent stem cells (iPSCs) from a mouse strain that expressed a green fluorescent protein. They then used skin cells from these mice to make iPSCs.

Japanese stem cell researcher Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs. To make iPSCs, adult cells are genetically engineered with four different genes and these genes de-differentiate the adult cells to a pluripotent stem cells state.

The iPSCs made from the green fluorescent mice were then differentiated into CPCs. They were able to isolate and identify CPCs by means of capturing all the cells that made Flt1 and Flt4.

According to Schenke-Layland, “Using our newly established cell surface markers, we could detect and isolate the Flt1- and Flt4-positive CPCs in culture. When we cultured the isolated mouse CPCs then in vitro, they actually developed – as well as the embryonic stem cell-derived progenitor cells – into endothelial cells, smooth muscle cells and more interestingly into functional heart muscle cells.”

To determine if these iPSC-derived CPCs could integrate into a living heart, they injected them into the hearts of living mice. 28 days later, the noticed that the injected hearts were loaded with green fluorescent cells that had differentiated into beating heart muscle that were fully integrated into the heart muscle tissue of the heart.

The next step is to determine if these CPCs can help heal a heart after a heart attack. Bone marrow-derived stem cells have been used to help heal the hearts of heart attack patients, and to date, these stem cells are safe, but only seem to help most people just little, even though they seem to help some patients more than others. However, iPSC-derived CPCs could potentially heal the heart to a greater degree.

According to Schenke-Layland, “We are currently focusing on research with human iPS cells. If we can show that cardiovascular progenitor cells can be derived from human iPS cells that have the ability to mature into functional heart muscle, we will have discovered a truly therapeutic solution for heart attack patients.”

See “Characterization and Therapeutic Potential of INduced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells;” Ali Nasar et al: PLoS ONE, 2012; 7 (10): e45603 DOI: 10.1371/journal.pone.0045603.

Maintaining the Reservoir of Blood Cell-Making Bone Marrow Stem Cells

Stowers Institute for Medical Research fellow Linheng Li has discovered a mechanism by which blood cell-making stem cells maintain a reservoir of cells that can replenish these stem cells under stressful conditions. His paper greatly advances our knowledge of these stem cells.

Li and his colleagues have identified two cell surface molecules that keep mouse hematopoietic stem cells from proliferating when they are not needed. Not all adult stem cells are created equally. Some are busy repairing damaged tissues while others held in reserve to replenish those stem cells that have worn themselves out healing other tissues. Blood cell-making stem cells or hematopoietic stem cells are no different. some are held in reserve while others are actively making new blood cells. What tells some to grow and others to lay low?

Li’s group found that two cell surface proteins known as Flamingo and Frizzled8 regulate hematopoietic stem cell (HSC) proliferation. The activity of these two proteins helps maintain a reserve pool of HSCs in mouse bone marrow. In doing so, they help control the delicate balance between long-term maintenance of stem cell populations in the bone marrow, and the requirements of ongoing tissue maintenance and regeneration.

Li explains: “HSCs daily produce billions of blood cells via a strict hierarchy of lineage-specific progenitors. Identifying the molecular signals that allow HSC populations to sustain this level of output over a lifetime is fundamental to understanding the development of different cell types, the nature of tumor formation, and the aging process. My hope is that these insights will help scientists progress towards new therapies for diseases of the blood.”

Currently, work in Li’s laboratory and other labs has provided a working model that predicts that HSCs consist of a population that only divides a few times a year and sit, essentially, in reserve. These reserve cells only become activated when they need to replace other active HSCs that have been damaged by the daily war-and-tear or in response to injury or disease that greatly depletes the blood supply (Li J. Exp Hematol. 2011;39(5):511-20 & Ezoe S, et al., Cell Cycle. 2004;3(3):314-8). Missing from this working model is how the reserve and active populations of HSCs are maintained and regulated.

Both of these HSC populations exist in specialized locations within the bone and these locations or “niches” provide external cues to regulate the proliferation of the respective HSC population. Approximately 90% of all HSCs frequently divide and are found in the central marrow near blood vessels and endothelial and perivascular cells. Reserve HSCs tend to be located in the spongy part of the bone (trabecular bone) at the end of long bones. Reserve HSCs are usually very intimately associated with immature versions of bone-making cells (pre-osteoblasts). In fact, the reserve HSCs tend to be in contact with the pre-osteoblasts and this contact is extremely important from a regulatory perspective.

In Li’s lab, graduate student Ryohichi Sugimura examined Flamingo and Frizzled8 on the surface of reserve HSCs. Both of these molecules participate in a signaling pathway known as the Wnt pathway, except that Wnt signaling normally occurs through a pathway known as the canonical Wnt pathway, but Wnt signaling can also occur though so-called non-canonical Wnt signaling pathways. Canonical Wnt signaling involves a secreted glycoprotein called the Wnt, which binds to a receptor. The receptor is a member of the Frizzled gene family and Frizzled binding to Wnt initiates a signaling cascade that culminates in the accumulation of a protein called beta-catenin in the nucleus. Beta-catenin associates with members of the TCF/LEF gene family to change gene expression in the cell. Noncanonical Wnt signaling occurs through changes in Calcium ion concentrations in the cell that elicit changes in cell behavior (see Rao TP, Kühl M Circ Res. 2010;106(12):1798-806).

Experiments in culture showed that reserve HSCs not only are in contact with pre-osteoblasts, but that the Flamingo protein accumulates at the interface between the HSC and the pre-osteoblast. Furthermore, Sugimura and his colleagues showed that Flamingo regulated the distribution of Frizzled8 in the HSC membrane. Other clues were also very curious. Members of the canonical Wnt signaling pathway in reserve HSCs were quite low, but components of the noncanonical Wnt signaling pathway were expressed at high levels in reserve HSCs.

Sugimura explained the significance of these data this way: “These observations indicated that the osteoblast niche provides a microenvironmentr in which non-canonical Wnt signaling prevails over canonical Wnt signaling under normal conditions. It also suggested that Flamingo and Frizzled8 may play a direct role in maintaining the pool of quiescent HSCs.”

Surgimura and his colleagues engineered mice that lacked the Flamingo and Frizzled8 proteins and they found that these mice had very few reserve HSCs. Additionally, HSC function was greatly reduced (>70%).

Treatment of normal mice with the drug 5-fluorouracil, which destroys dividing HSCs, confirmed the role of non-canonical Wnt signaling in the maintenance of reserve HSCs. Under these conditions, noncanonical Wnt signaling components decreased and canonical Wnt signaling components increased as the reserve HSCs transitioned to frequently-dividing HSCs.

Li concluded: “A better understanding of how the balance shifts between the two will provide the necessary mechanistic insight that allows us to reduce non-canonical Wnt-signaling and dial-up canonical Wnt signaling in order to activate quiescent HSCs during aging. But the knob will have to be turned carefully. If the balance shift too far in favor of canonical Wnt signaling, it may well increase the risk of leukemia.”