Stem Cell-like Megakaryocyte Progenitors Replenish Platelets After Inflammatory Episodes


A paper that appeared in the journal Cell Stem Cell from the laboratory of Marieke A.G. Esters, from the Heidelberg Institute for Stem Cell Technology and Experimental Medicine in Heidelberg, Germany has answered a long-standing question about how our bodies regenerate platelets using so many of them.

When we suffer damage to our bodies from surgery, accidents, infections, or other physical insults, we tend to use a lot of platelets. Platelets are small cells in the blood that help the blood clot once we cut ourselves. Platelets, however, take some time to form. How then do we rapidly regenerate the platelet pool during such stressful conditions?

Esters and her team have shown that the bone marrow contains stem-like cell called a “single-lineage megakaryocyte-committed progenitor” or SL-MkPs. Platelets bud from a large cell called a “megakaryocyte,” and megakaryocytes form from the hematopoietic stem cells that reside in the bone marrow. Hematopoietic stem cells make all the blood cell that course through our blood vessels and continue to replace those cells throughout our lifetime. Hematopoietic stem cells personify what it means to be a multipotent stem cell.

Haas et al, graphical abstract 5.5x5.5

This newly-identified stem cell population, the SL-MkP actually shares many features with multipotent hematopoietic stem cells and provides a stem cell population that is lineage-restricted (that means they can only form one type of cell) for emergency purposes.

Normally, SL-MkPs are maintained in an inactive, almost sleep-like state. In this state, SL-MkPs do not contribute very much to making platelets in the blood. There is some gene expression in this sleepy state, but protein synthesis is turned way down.

In response to acute inflammation, SL-MkPs wake up and become activated. Upon activation, these cells ramp up protein synthesis and mature into full-blown SL-MkPs that efficiently replenishment of platelets that are lost during high levels of inflammation. Thus, there is an emergency system that accommodates platelet depletion during acute inflammation and replenishes the platelet pool.

Making Platelets in the Culture Dish


Bone marrow-based cells known as megakaryocytes are rather uncommon in bone marrow, but these cells are very important for the health and daily operation of the human body. Megakaryocytes, you see, produce platelets, which are critical to clotting broken blood vessels and wound healing. Generating megakaryocytes in cell culture has proven to be rather difficult, but induced pluripotent stem cells might provide a way to make megakaryocytes in culture.

Megakaryocyte
Megakaryocyte

Platelets_large

The differentiation of induced pluripotent stem cells (iPSCs) into megakaryocytes could potentially create a renewable cell source of platelets for treating patient with “thrombocytopenia,” which is a deficiency of platelets. Zack Wang and his colleagues from Johns Hopkins University in Baltimore, Maryland have developed a protocol to make megakaryocytes in culture from iPSCs. However, more than that, Wang and his co-workers wanted to make patient-specific platelets in culture without using any animal products and with compounds that were approved by the US Food and Drug Association. Such a protocol would demonstrate that using such cells in human patients is feasible and safe.

Wang and his colleagues developed an efficient system that generated megakaryocytes from human iPSCs without the use of animal feeder cells and without animal products (known as xeno-free condition). Several crucial reagents necessary to differentiate iPSCs into megakaryocytes into were replaced with Food and Drug Administration-approved pharmacological reagents that included romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). Wang and his group used their method to induce megakaryocytes generation from human iPSCs derived from 23 individuals in two steps: 1) generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and 2) generation and expansion of CD41+CD42a+ megakaryocytes from HPCs for an additional 5 days. After 19 days, Wang and his group observed abundant CD41+CD42a+ megakaryocytes that also expressed the megakaryocyte-specific cell-surface proteins CD42b and CD61. These cells were also polyploid, which means that they had multiple copies of each chromosome rather than just 2 copies (≥16% of derived cells with DNA contents >4N). Gene expression studies showed that megakaryocytic-related genes were highly expressed in their cultured megakaryocytes.

Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.
Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.

This protocol could be used to further understand the medical conditions that lead to thrombocytopenia. Deeper understanding of these medical conditions will hopefully lead to better treatments of them. Also, Wang’s protocol may lead to the generation of large numbers of platelets in culture that could then be given to patients who need them.

Stem Cells to Make Red Blood Cells and Platelets in Culture


A collaborative study between Boston University School of Public Health and researchers at Boston Medical Center has used induced pluripotent stem cells to make unlimited numbers of human red blood cells and platelets in culture.

This finding could potentially reduce the need for blood donations to treat patients who require blood transfusions. Such research could also help researchers examine fresh and new therapeutic targets in order to treat blood diseases such a sickle-cell anemia.

The lead scientist on this project was George Murphy, assistant professor of medicine at Boston University School of Medicine and co-director of the Center for Regenerative Medicine at Boston University. Murphy’s main collaborator was David Sherr, professor of environmental health at Boston University School of Medicine and the Boston University School of Public Health.

Induced pluripotent stem cells or iPSCs are made from adult cells by applying genetic engineering technology to the adult cells that introduces genes into them. The introduction of four specific genes de-differentiates the adult cells into pluripotent stem cells that can, potentially, differentiate into any adult cell type. This makes iPSCs powerful tools for research and potential therapeutic agents for regenerative medicine.

In this study, Murphy and others used iPSCs from the CreM iPS Cell Bank and exposed them to a battery of different growth factors in order to push them to differentiate into different adult cell types. They were looking for the precise cocktail to differentiate iPSCs into red blood cells, since they wanted to further study red blood cell development in detail.

One group of compounds given to the set of iPSCs were molecules that activate “aryl hydrocarbon” receptors. Aryl hydrocarbon receptors (AHRs) play important roles in the expansion of hematopoietic stem cells, which make blood cells, since antagonism of AHRs promotes expansion of hematopoietic stem cells (see AE Boitano et al.,Science 10 September 2010: Vol. 329 no. 5997 pp. 1345-1348). In this case, however, Murphy and his colleagues observed a dramatic increase in the production of functional red blood cells and platelets in a short period of time. THis suggests that the ARH is important for normal blood cell development.

Aryl Hydrocarbon Receptor
Aryl Hydrocarbon Receptor

“This finding has enabled us to overcome a major hurdle in terms of being able to produce enough of these cells to have a potential therapeutic impact both in the lab and, down the line, in patients,” said Murphy. “Additionally, our work suggests that AHR has a very important biological function in how blood cells form in the body.”

“Patient-specific red blood cells and platelets derived from iPSC cells, which would solve problems related to immunogenicity and contamination, could potentially be used therapeutically and decrease the anticipated shortage and the need for blood donation,” added Murphy.

iPS-derived cells have tremendous potential as model systems in which scientists can test and develop new treatments for disease, given that such diseases can be constructed in the laboratory. These iPSC-derived red blood cells could be used by malaria researchers, and IPSC-derived platelets could be used to explore cardiovascular disease and treatments for blood clotting disorders.

Because my mother died from myelodysplasia, this finding has some personal interest to me. Mom had a difficult blood type to match, since she had the Bombay blood type (H). Finding blood for her was a major tour de force, and as she received blood that was less and less well matched to her body, she suffered the ravages of poorly matched blood. A treatment of red blood cells made from IPSCs derived from her own cells might have extended her life and even improved her quality of life in her later years.

I look forward to this research eventually culminating in clinical trials.

ACT to Start Clinical Trial of iPSC-Derived Platelets


Platelets are blood cells that help clot our blood when blood vessels are damaged. They are small cells, and are only about 20% of the diameter of red blood cells. There are typically about 150,000-350,000 platelets per microliter of blood. Despite these large numbers, platelets only compose a tiny fraction of the blood volume. As mentioned, the main function of platelets is to prevent bleeding. Platelets Platelets are produced in the bone marrow by the typical process of blood cell production. Hematopoietic stem cells in the bone marrow divide to renew and form a progenitor cell that differentiates into either a pro-erythrocytes, which becomes a red blood cell, or a promegakaryocyte. Promegakaryoyctes differentiate into megakaryocytes, which are the cells that form platelets. Platelets bud off the cytoplasm of the megakaryocytes. Consequently, platelets do not possess a nucleus. Each megakaryocyte produces between 5,000 and 10,000 platelets. Platelet differentiation Megakaryocyte and platelet production is regulated by a hormone called thrombopoietin, which is produced by the liver and kidneys. The average lifespan of circulating platelets is 5-9 days, and older platelets are destroyed by cells in the spleen and by Kupffer cells in the liver that gobble up the old platelets and recycle their components. Many platelets are held in reserve by the spleen, which are released when needed by contraction of the spleen, which is induced by the sympathetic nervous system (fight or flight response).

The cells that compose the inner surface of blood vessels normally inhibit platelet activation by producing various molecules, such as nitric oxide, and prostaglandin I2. Blood vessel cells also make a cell surface molecule called von Willebrand factor, which helps them adhere to the cable-like protein collagen that lies outside the blood vessels. Injury to blood vessels reduces the production of these inhibitory molecules and exposes the platelets and blood to collagen and von Willebrand factor (vWF). When the platelets contact collagen or vWF, they become activated. This activation manifests itself is several ways. First of all, the platelets dump the granules that they store. These granules contain several important molecules, but they also place new surfaced proteins on the outside of the platelets that help them clump together. Activated platelets also change their shape to become more spherical, and extensions of the surface form. This gives them a kind of star-shape.

Activated_platelets,

Other molecules released from their granules include ADP, which is a platelet-activating molecule, the neurotransmitter serotonin, which induces blood vessels to constrict (this staunches blood loss), blood clotting factors (factors V and XIII for those who are interested), and some growth factors. Platelet activation also induces platelets to synthesize a molecule called thromboxane A2, which, like ADP, activates other platelets. Thus platelet activation is a self-amplifying event and gets more and more platelets involved in the act. I hope I have convinced you of the importance of platelets.

Some people have problems with insufficient numbers of platelets, and they have trouble properly clotting their blood.  Therefore, giving them more platelets is an excellent way to treat them, but a source of platelets must be found in order to give them to the patient. Enter the Massachusetts-based biotechnology company, ACT.  Advanced Cell Technologies from Marlborough, Mass., wants to test platelets made from reprogrammed cells; that is to say induced pluripotent stem cells.   Patients with some types of leukemia, anemia and other conditions need repeated infusions of platelets to avoid bleeding to death.  Additionally, the immune system of such patients can become sensitized to donated platelets, which compromises their effectiveness.

Platelets made from induced pluripotent stem cells (iPSCs) could overcome that problem because they were derived from a patient’s own cells. Platelets also seem to be the ideal cell for this technology because the platelets live for such s short time, they do not have a nucleus, and therefore, cannot cause tumors. Alan Michelson, a platelet researcher at Harvard Medical School and Boston Children’s Hospital who would lead a clinical trial in the U.S. studying ACT’s stem-cell derived platelets, said, “This would really be a dramatic advance in medicine, but it remains to be seen if this would be successful.”

Robert Lanza, ACT’s chief scientific officer, said that the company has the “capacity to make enough” platelets for the initial clinical trials but would “take time to scale up for widespread use.”  He added, “It doesn’t require any embryos. It doesn’t require eggs. It doesn’t require any destruction of embryos.” ACT has proposals for clinical trials and the company has said that testing could begin as early as the end of next year if regulators sign off.  The U.S. Food and Drug Administration has declined to comment, since federal rules prevent it from discussing any therapies that may be under development.

According to Lanza, the proposed U.S. trial would potentially infuse  normal platelets and stem-cell-derived platelets into eight patients and compare how well the cells functioned.  Because the platelets can be labeled, blood draws from the patients would determine if the iPSC-derived platelets behave like bona fide platelets.

According to Cynthia Dunbar, a stem-cell researcher at the National Heart, Lung and Blood Institute and editor of the journal Blood, if the iPSC-derived platelets worked like normal platelets, “the potential impact would be great.”