Stem-Cell Gene Therapy for Sickle Cell Disease


Donald Kohn, a professor of pediatrics and microbiology, immunology and molecular genetics in the UCLA College of Letters and Science, and his colleagues, have successfully established the means to cure sickle-cell disease. This strategy uses hematopoietic (blood-producing) stem cells from the bone marrow of patients with sickle-cell disease in order to treat the disease itself.

This approach provides a revolutionary alternative to current treatments, since it creates self-renewing, normal blood cells by inserting a gene that abrogates the sickling properties into hematopoietic stem cells. With this technique, there is no need to identify a matched donor, and therefore, patients avoid the risk of their bodies rejecting donor cells.

During the clinical trial, the anti-sickling hematopoietic stem cells will be transplanted back into patients’ bone marrow to increase the population of “corrected” cells that make red blood cells that don’t sickle. Kohn will hopefully begin enrolling patients in the trial within three months. The first subject will be enrolled and observed for safety for six months. The second subject will then be enrolled and observed for safety for three months. If evaluations show that no problems have arisen, the study will continue with two more subjects and another evaluation, until a total of six subjects have been enrolled.

Sickle cell disease, which affects more than 90,000 individuals in the U.S., is seen primarily in people of sub-Saharan African descent. It is caused by an inherited mutation in the beta-globin gene that transforms normal-shaped red blood cells, which are round and pliable, into rigid, sickle-shaped cells. Normal red blood cells are able to pass easily through the tiniest blood vessels (capillaries) and carry oxygen to organs like the lungs, liver and kidneys. However, sickled cells get stuck in the capillaries, depriving the organs of oxygen, which can lead to organ dysfunction and failure.

Current treatments include transplanting patients with hematopoietic stem cells from a donor. This is a potential cure for the disease, but due to the serious risks of rejection, only a small number of patients have undergone this procedure, and it is usually restricted to children with severe symptoms.

“Patients with sickle-cell disease have had few therapeutic options,” Kohn said. “With this award, we will initiate a clinical trial that we hope will become a treatment for patients with this devastating disease.”

Finding for this work comes from new grants to researchers at UCLA’s Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, which total nearly $21 million.  These grants were announced Dec. 12 at a meeting of the California Institute of Regenerative Medicine (CIRM) Citizen’s Oversight Committee.  They are apart of the state agency’s Disease Team Therapy Development III initiative.

Stress-Resistant Stem Cells From Fat


During liposuction patients lose a fat cells, fat-based mesenchymal stem cells, and now, according to new results from UCLA scientists, stress-enduring stem cells.

This new stem cell population has been called a Multi-lineage Stress-Enduring Adipose Tissue or Muse-AT stem cells. UCLA scientists found Muse-AT stem cells by accident when a particular machine in the laboratory malfunctioned, killing all the cells found in cells from human liposuction, with the exception on the Muse-AT stem cells.

Gregorio Chazenbalk from the UCLA Department of Obstetrics and Gynecology and his research team discovered, after further tests on Muse-AT stem cells, that they not only survive stress, but might be activated by it.

The removal of Muse-AT stem cells from the human body by means of liposuction revealed cells that express several embryonic stem cell-specific proteins (SSEA3, TR-1-60, Oct3/4, Nanog and Sox2). Furthermore, Muse-AT stem cells were able to differentiate into muscle, bone, fat, heart muscle, liver, and neuronal cells. Finally, when Chazenbalk and his group examined the properties of Muse-AT stem cells, they discovered that these stem cells could repair and regenerate tissues when transplanted back into the body after having been exposed to cellular stress.

Muse-ATs express pluripotent stem cell markers. Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X. doi:10.1371/journal.pone.0064752.g002
Muse-ATs express pluripotent stem cell markers.
Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.
doi:10.1371/journal.pone.0064752.g002
“This population of cells lies dormant in the fat tissue until it is subjected to very harsh conditions. These cells can survive in conditions in which usually cancer cells can survive. Upon further investigation and clinical trials, these cells could prove a revolutionary treatment option for numerous diseases, including heart disease, stroke and for tissue damage and neural regeneration,” said Chazenbalk.

Purifying and isolating Muse-AT stem cells does not require the use of a cell sorter or other specialized, high-tech machinery. Muse-AT stem cell can grow in liquid suspension, where they grow as small spheres or as adherent cells that pile on top of each other to form aggregates, which is rather similar to embryonic stem cells and the embryoid bodies that they form.

Isolation and morphologic characterization of Muse-ATs. (A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity. doi:10.1371/journal.pone.0064752.g001
Isolation and morphologic characterization of Muse-ATs.
(A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.
doi:10.1371/journal.pone.0064752.g001

We have been able to isolate these cells using a simple and efficient method that takes about six hours from the time the fat tissue is harvested,” said Chazenbalk. “This research offers a new and exciting source of fat stem cells with pluripotent characteristics, as well as a new method for quickly isolating them. These cells also appear to be more primitive than the average fat stem cells, making them potentially superior sources for regenerative medicine.”

Embryonic stem cells and induced pluripotent stem cells are the two main sources of pluripotent stem cells. However, both of these stem cells have an uncontrolled capacity for differentiation and proliferation, which leads to the formation of undesirable teratomas, which are benign tumors that can become teratocarcinomas, which are malignant tumors. According to Chazenbalk, little progress has been made in resolving this defect (I think he overstates this).

Muse-AT stem cells were discovered by a research group at Tokohu University in Japan and were isolated from skin and bone marrow rather than fat (see Tsuchiyama K, et al., J Invest Dermatol. 2013 Apr 5. doi: 10.1038/jid.2013.172). The Japanese group showed that Muse-AT stem cells do not form tumors in laboratory animals. The UCLA group was also unable to get Muse-AT stem cells to form tumors in laboratory animals, but more work is necessary to firmly establish that these neither form tumors nor enhance the formation of other tumors already present in the body.

Chazenbalk also thought that Muse-AT stem cells could provide an excellent model system for studying the effects of cellular stress and how cancer cells survive and withstand high levels of cellular stress.

Chazenbalk is understandable excited about his work, but other stem cells scientists remain skeptical that this stem cells population has the plasticity reported or that these cells are as easily isolated as Chazenbalk says.  For a more skeptical take on this paper, see here.