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Diabetes (otherwise known by the scientific name diabetes mellitus) is a complicated condition and there are many different types of diabetes. Here we’ll take you through everything you need to know. What is diabetes? Diabetes is a serious condition where your blood glucose level is too high. It can…

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In this activity, you will work in pairs or in a group to make a short presentation (maximum 8 min) to the class on the use of stem cells as a treatment for ONE disease. The presentation must be supported by scientific evidence that must be appropriately referenced. Therefore, check all the resources provided in the corresponding column of your disease to focus your research on.

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You must upload your presentation under the corresponding column of your disease.

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While your peers are presenting, you must complete a table with relevant information

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Create a new document and edit with others at the same time -- from your computer, phone or tablet. Get stuff done with or without an internet connection. Use Docs to edit Word files. Free from Google.

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Autologous Stem Cell Transplant | Animation Video

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Over the past several decades, researchers have identified hundreds of genes that contribute to inherited eye diseases, including Stargardt disease. This information has led to better diagnostic tests, and is providing insight into possible treatments.

Scientists are also working to find new mutations in the ABCA4 gene, and in other genes, that might contribute to Stargardt disease. NEI's National Ophthalmic Disease Genotyping and Phenotyping Network () is an important resource for these efforts. The eyeGENE network gives patients access to genetic studies and clinical trials, while giving researchers access to patient DNA samples and clinical information.

Many studies continue to explore the biology and genetics of Stargardt disease, and of macular degeneration more generally. For example, NEI is conducting a natural history study of Stargardt and other ABCA4-related diseases. The study is following 45 individuals with ABCA4 mutations for five years. The main goals are to better understand the natural course of the disease, to make contact with people who may be interested in future clinical trials, and to collect blood, skin, and DNA samples from those people. These samples can be studied in the lab to explore the mechanisms of Stargardt disease.

Such mechanistic studies can lead to new treatment strategies. One strategy currently under study is to reduce the build-up of lipofuscin and other toxic byproducts in the retina. An NEI-funded group based at Columbia University is working with a synthetic form of vitamin A, called ALK-001, that isn't readily converted into lipofuscin. In mice and larger animal models, an oral form of ALK-001 slows the formation of lipofuscin deposits. ALK-001 is being tested for safety in healthy volunteers before testing begins on people with Stargardt disease.

A group based at Case Western Reserve University and supported by the NEI Translational Research Program on Therapy for Visual Disorders is taking another approach. The group has tested a panel of drugs already deemed safe by the Food and Drug Administration (FDA) in a mouse model of Stargardt. They found that some FDA-approved drugs can reduce retinal damage in the mice. They are now evaluating chemical compounds with a similar structure to FDA-approved drugs, and exploring different modes of drug delivery.

Gene therapy - that is, repairing or replacing the defective ABCA4 gene - also holds promise for treating Stargardt disease. Gene replacement therapy requires a method for delivering the gene of interest into cells, and for some diseases, the solution has been to package the gene inside a small, harmless virus called the adeno-associated virus (AAV). The ABCA4 gene is too large to fit within this virus. Researchers funded by NEI therefore modified a larger virus from the lentivirus family, and engineered it to carry ABCA4. Tests in the Stargardt mouse model showed that this approach can reduce lipofuscin accumulation. Oxford Biomedica has refined this technology, and licensed it under the name StarGen. Human safety trials of StarGen began in 2011.

Finally, stem cell-based therapies are showing promise for Stargardt disease in clinical trials. Stem cells are immature cells that can generate many mature cells types in the body, including the photoreceptors that die off in Stargardt disease. Human stem cells can be derived from embryonic or adult tissues. Both kinds of cells are being tested in patients with Stargardt disease and age-related macular degeneration (AMD), which is a leading cause of vision loss in the United States.

A U.S. company called Advanced Cell Technology (ACT) is conducting a trial of retinal pigment epithelium (RPE) cells for AMD and Stargardt disease. These cells provide support and nourishment to the retina. The RPE cells under study in the ACT trial are derived from human embryonic stem cells.

Other clinical and laboratory studies are making use of adult cells that have been reprogrammed into stem cells, called induced pluripotent - or iPS - cells. In 2014, Japanese scientists launched the first clinical trial of iPS cells to treat AMD, and indeed the first trial ever of iPS cells. The goal is to coax the iPS cells into making RPE cells. NEI scientists are planning a similar trial and going a step further - by seeding the RPE cells onto a scaffold so that they form a sheet, similar to how they arrange themselves in the eye. NEI scientists also plan to make iPS cells from the skin cells collected in the Stargardt natural history study. These iPS cells could be used to make photoreceptors and RPE cells - for use in potential cell therapies and as a research tool to study how potential drugs will affect these cell types.

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Stargardt disease is an inherited disorder that usually causes vision loss in childhood or adolescence. It is also called Stargardt macular dystrophy, juvenile macular degeneration, or fundus flavimaculatus. Learn about the symptoms, causes, diagnosis, and treatment of Stargardt disease and current research.

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Abstract

Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic β cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy.

Results

Generation of iPS Cells from Patients with T1D.

Skin biopsies were obtained from two Caucasian males with T1D of 11- and 27-years duration, respectively. Patient 1 was presented at age 21 with polyuria, polydypsia, and a blood glucose concentration of 680 mg/dL. Patient 2 was presented at age 3 in diabetic ketoacidosis requiring hospitalization. For both individuals, the body mass index did not exceed 22 or 23, respectively. HLA haplotypes, and other clinical data, are given in Table 1. Fibroblasts obtained from skin biopsies were cultured and infected with a combination of retroviruses encoding the transcription factors OCT4, SOX2, and KLF4 ( 15). Starting 4 weeks after infections, colonies were picked based on their morphological resemblance to human ES cell colonies and expanded. To test whether the cell lines expressed pluripotency markers, we verified the presence of alkaline phosphatase (AP) activity, as well as staining for antibodies to OCT4, NANOG, SOX2, TRA1-60, TRA1-81, and SSEA4. The reprogrammed cells were positive for AP activity, and were reactive to antibodies against all pluripotency markers tested ( Fig. 1 A and B). Karyotype analysis and DNA fingerprinting verified that the T1D patient-specific DiPS lines were generated from the parental fibroblast line and maintained a normal karyotype ( Fig. S1 and Table S1).

The analysis of pluripotency markers was extended to include expression of the endogenous genes encoding NANOG, OCT4, TERT, REX1, SOX2, and GDF3 by semiquantitative PCR analysis. Expression of these genes in the DiPS lines was similar to human ES cells, but was absent in the parental fibroblasts ( Fig. 2 A). As previously described, KLF4 is expressed in fibroblasts, as well as in human ES cells and DiPS cells ( Fig. 2 A) ( 15). Expression of β-ACTIN was used as a control for RNA recovery and to allow semiquantitative comparison of expression levels. Omission of the reverse transcription reaction was used as a control for specificity and gave no bands of expected size in the semiquantitative PCRs ( Fig. 2 A). A more global view of the gene expression profiles for the parental fibroblasts, DiPS, and hES lines, was obtained using DNA microarrays. Hierarchical clustering revealed that DiPS cell lines from both patients were highly similar to the human ES cell lines HUES4, HUES6, and HUES8 ( 17), while exhibiting low similarity to fibroblasts ( Fig. 2 B). The coefficient of determination (r ² = square of the correlation coefficient) was 0.72-0.74 for DiPS compared with parental fibroblasts, and 0.94-0.98 for DiPS compared with HUES cells ( Table S2). We conclude that DiPS cells closely resemble human ES cells in global gene expression as we have described for iPS from primary human cells before ( 18).

To determine whether the viral transgenes were silenced, we performed exogenous gene-specific quantitative PCR analysis. Compared with the infected fibroblast control, the transgene expression in the DiPS cells was low or at background levels ( Fig. 2 C), presumably due to anticipated viral gene silencing ( 15). We conclude that the DiPS lines closely resemble human ES cells in their expression profiles and, like other reported iPS cells, have silenced the transgenes.

DiPS Cells Spontaneously Differentiate into Cell Types of Different Germ Layers.

DiPS cells were allowed to spontaneously differentiate in embryoid body (EB) cultures. EB formation was achieved by culturing DiPS cells in differentiation media on low-attachment plates, followed by plating onto gelatin-coated dishes for additional culture. We analyzed the morphologically differentiated cells for expression of markers for the endodermal (SOX17 and FOXA2), mesodermal (SMA), and ectodermal (TUJ1) lineages in differentiated cultures ( Fig. 3 A). The differentiated DiPS cells were found positive for cells of all three germ layers. Also, we verified pluripotency of DiPS cells in teratoma formation assays. After injection of DiPS cells into immunocompromised mice, DiPS cells formed teratomas containing derivatives of endoderm (glandular structures), mesoderm (cartilage), and ectoderm (nerve fibers, pigmented epithelium, and melanocytes) ( Fig. 3 B). We conclude that patient-specific DiPS cells can spontaneously differentiate into derivatives of all three germ layers.

DiPS Cells Can Be Differentiated Along the Endodermal/Pancreatic Lineage.

We applied a directed differentiation protocol to the DiPS cells to determine whether they can be differentiated toward an insulin producing/glucose responsive cell. The protocol followed a stepwise differentiation protocol that relies on the generation of intermediate precursors thought to be similar to populations present in the developing embryo. DiPS cells were subjected to a protocol that directs differentiation to definitive endoderm followed by gut tube endoderm before further differentiation into pancreatic progenitors, and finally, the β-like cell ( Fig. 4 A). As determined by immunofluorescence analysis for SOX17 and FOXA2 expression, DiPS lines from both patients could respond to WNT3A and Activin A treatment to differentiate into definitive endoderm, similar to human ES cells ( Fig. 4 B Upper). Further differentiation toward gut tube endoderm (HNF4a and HNF1b positive), and pancreatic progenitors (PDX1 and HNF6 positive) was achieved by supplying FGF10 and cyclopamine, and FGF10, cyclopamine, retinoic acid, and (−)-Indolactam V, respectively. FOXA2 expression was detected in both definitive endoderm and pancreatic progenitors ( Fig. 4 B) as expected from normal embryonic development ( 19). The expression of these transcription factors was validated by semiquantitative PCR ( Fig. 4 C).

Further differentiation of DiPS-derived pancreatic progenitors toward the endocrine lineage yielded cells that were positive for somatostatin, glucagon, and insulin ( Fig. 5 A). Insulin-positive cells were C-peptide positive, excluding insulin uptake from the media. Semiquantitative PCR analysis confirmed the expression of endocrine-specific gene products of INSULIN, PDX1, NKX2.2, GLUCAGON, and SOMATOSTATIN ( Fig. 5 B). To address whether the insulin could be released on glucose stimulation in vitro, we exposed the DiPS-derived insulin producing cells to low or high concentrations of glucose. The DiPS-derived population released human C-peptide on glucose stimulation ( Fig. 5 C). The amount of released C-peptide after high (20 mM) glucose stimulation was at least 5-fold higher than after low (2.5 mM) glucose stimulation. We conclude that DiPS cells can be differentiated to insulin producing/glucose-responsive cells.

Discussion

Insight into the pathogenesis of T1D comes largely from rodent models such as the NOD mouse or the BB rat. However, the existing rodent models are not fully representative of the relevant processes in patients with T1D. Indeed, insights from rodent models have frequently not translated well to the clinic ( 1- 3). Access to genetically-predisposed human cells whose biological status ultimately defines the disease enables previously impossible mechanistic studies in vitro, and in vivo transplant systems. We have generated human PS cells from two patients with T1D. These DiPS cells provide a starting material for patient-specific disease modeling and for testing differentiation protocols. In mice, inclusion of c-Myc as a fourth reprogramming factor is associated with lethal tumor formation in contrast to reprogramming with three factors (Oct4, Sox2, and Klf4) ( 20). The reprogramming process described here was achieved by using three factors (OCT4, SOX2, and KLF4), omitting the oncogene C-MYC. Consistent with previously described iPS cell lines, transgene silencing was observed in DiPS cells ( 4, 15, 21, 22). Induced PS cells can be generated from mouse cells without permanent or transient integration of the transforming factors in the genome ( 23, 24), and this approach has recently extended to human cells ( 25). Currently, generation of human iPS cells involves delivery of DNA in a manner that allows potential integration into the genome, but alternative approaches are likely to be available in the near future ( 26). In any event, insufficient characterization of the reprogramming process and its product precludes use of iPS cells in cell replacement therapy at this point. However, the generation of patient-specific PS cells paired with differentiation into cell types relevant to the disease promises to provide valuable insights into disease pathogenesis. The DiPS cells described here are pluripotent based on similarities in gene expression to human ES cells and their ability to spontaneously differentiate into cells of different germ layers. Notably, we have differentiated these patient-specific DiPS cell lines to a cell population relevant to T1D, an insulin-producing and glucose-responsive β-like cell. Previously, iPS cells generated from foreskin fibroblasts, but not from diabetes patients, using four factor reprogramming (OCT4, SOX2, KLF4, and C-MYC) were shown to differentiate to insulin-producing clusters ( 27), leaving open the task of generating patient-specific DiPS, and testing their potential to differentiate toward insulin-producing β-like cells. Although DiPS cells can be differentiated to insulin producing cells, the efficiency of this process is low, possibly because the differentiation protocol has not been optimized or due to variation in the differentiation propensities of human PS cells ( 28). Interestingly, we observed differences between DiPS lines from the same patient, potentially due to transgene reactivation or incomplete silencing. As a consequence, characterization of multiple lines and future efforts to generate DiPS cells without viral integration will help address this issue.

As observed with human ES cells, β-like cells derived from DiPS are glucose responsive, but until the differentiation protocols are improved, it is not yet possible to directly compare these cells with purified pancreatic β cells. In addition to variation in differentiation propensities, potential deviations in T1D disease onset and progression will require the generation of multiple DiPS lines to reflect the human population afflicted with T1D. The two cell lines from different patients described here represent a starting point for this larger task.

Differentiation of DiPS cells to β-like cells is relevant not only for the long term possibility of autologous cell replacement therapy, but also for disease modeling. We propose to extend this investigation to generate cell types of the immune system that may allow the generation of a cellular interaction model of T1D. This approach should provide a way to investigate T1D disease onset and progression in vitro and/or in reconstituted animal models. These in vitro and in vivo systems will also be useful for testing of preventative and therapeutic strategies.

Materials and Methods

Cell Culture.

Skin fibroblasts from T1D patients and controls were derived from explants of 3-mm dermal biopsies after informed consent under protocols approved both by Harvard University and Columbia University College of Physicians and Surgeons. Briefly, 3-mm skin biopsies were minced with scalpels into smaller pieces, and tissue fragments were placed into a 60-mm tissue culture dish under a sterile coverslip held down by sterilized silicon grease under one corner. Media was added to completely immerse the coverslip, and dishes were incubated at 37 °C in a humidified incubator (5% CO ₂). Media was changed every 5 days without disturbing the coverslip. Fibroblasts grew out of the tissue fragments, and when sufficiently numerous, cells were trypsinized and expanded. Subsequently, fibroblasts were maintained in fibroblast medium (DMEM supplemented with 10% FBS, glutamine, sodium pyruvate, nonessential amino acids, and penicillin/streptomycin). The resulting fibroblasts lines are referred to as fibroblast Harvard (H) lines 1 and 2.

Human ES cell and DiPS lines were cultured in human ES media (knockout DMEM supplemented with 10% knockout serum replacement, 10% human plasma fraction, 10 ng/mL bFGF, nonessential amino acids, β-mercaptoethanol, L-glutamine, and penicillin/streptomycin). Cultures were maintained on mouse embryonic fibroblast feeders and passaged enzymatically using either 0.05% Trypsin (GIBCO) or Collagenase type IV.

Reprogramming.

VSVG-coated retroviruses were generated according to standard procedures. One day before infection, 10E5 fibroblasts were seeded per well of a six well plate. Fibroblasts were infected on days 1 and 2 with a combination of OCT4, SOX2, and KLF4 containing Moloney viruses (constructs were obtained from Addgene) ( 15). The media was changed on day 3 to DMEM supplemented with 10% FBS, L-glutamine, penicillin/streptomycin, nonessential amino acids, and sodium pyruvate. A day later the cells were split onto gelatinized 10-cm cell culture dishes. Subsequently, the cells were fed every other day with human ES cell media. Generation of DiPS lines H2.4, H2.3, and H2.1b occurred with supplementation of the media with 1 mM valproic acid during the reprogramming process as described before ( 18), whereas DiPS lines H2.1, H1.1, H1.5 were generated without addition of the chemical. Colonies were picked starting ≈4 weeks after infection.

Spontaneous Differentiation.

Spontaneous differentiation through EB formation was initiated by dissociation of human DiPS cells using collagenase IV treatment, and subsequent transfer to low attachment 6-well plates in knockout DMEM supplemented with 20% knockout serum replacement, nonessential amino acid, β-mercaptoethanol, L-glutamine, and penicillin/streptomycin. After 8-10 days suspension culture, EBs were transferred to gelatin-coated plates and cultured for an additional 8-10 days as attachment culture.

For teratoma formation assays DiPS cells were collected by collagenase IV treatment, and injected s.c. into immunocompromised mice (NOD-SCID or SCID-Beige mice). Teratomas were collected 7-10 weeks after injection, and processed according to standard procedures for paraffin embedding and hematoxylin and eosin staining.

Directed Differentiation.

Directed differentiation was conducted as described ( 13, 29) with the following modifications: human DiPS cells were cultured on MEF feeder cells to 70-80% confluency, then treated with 25 ng/mL WNT3A (R&D systems) + 100 ng/mL Activin A (R&D systems) in advanced RPMI (A-RPMI; Invitrogen) supplemented with 1×L-Glu and 1×PS for 1 day, followed by treatment with 100 ng/mL Activin A in A-RPMI supplemented with 1×L-Glu, 1×PS and 0.2% FBS (Invitrogen). Two days later, the media was changed to 50 ng/mL FGF10 (R&D systems) + 0.25 μM KAAD-CYC (Calbiochem) in A-RPMI supplemented with 1×L-Glu, 1×PS and 2% FBS and maintained for additional 2 days. Cells were then transferred to 50 ng/mL FGF10 + 0.25 μM KAAD-CYC + 2 μM RA (Sigma) in DMEM supplemented with 1×L-Glu, 1×PS, 1× B27 (Invitrogen) and cultured for an additional 4 days. The media was then changed to 50 ng/mL FGF10 + 300 nM ILV (Axxora) in DMEM supplemented with 1×L-Glu, 1×PS, 1× B27 and cultured for an additional 4 days. Then, cells were transferred to 50 ng/mL EX-4 (Sigma) + 10 μM DAPT (Sigma) in DMEM supplemented with 1×L-Glu, 1×PS, 1× B27 and cultured for an additional 6 days. Cells were then cultured in 50 ng/mL HGF (R&D systems) + 50 ng/mL IGF1 (R&D systems) in CMRL-1066 (Invitrogen) supplemented with 1×L-Glu, 1×PS, 1× B27 for 6 days.

Immunofluorescence.

Immunofluorescence staining was performed using primary antibodies against C-peptide (4020-01; Linco), FOXA2 (07-633; Upstate), glucacon (4031; Linco), HNF6 (sc-13050; Santa Cruz Biotechnology), insulin (A0564; Dako), NANOG (ab21624; Abcam), NKX2.5 (sc-14033; Santa Cruz Biotechnology), OCT4 (sc-5279; Santa Cruz Biotechnology), PDX1 (AF2419; R&D systems), SMA (A5228; Sigma), somatostatin (A0566; Dako), SOX2 (sc-17320; Santa Cruz Biotechnology), SOX17 (AF1924; R&D systems), SSEA4 (MAB4304; Chemicon), TRA-1-60 (MAB4360; Chemicon), TRA-1-81 (MAB4381; Chemicon), and TUJ-1 (MMS-435P; Covance Research Products). Appropriate secondary antibodies were obtained from Molecular Probes.

Gene Expression Analysis.

RNA was isolated from cells using RNAeasy kit (Qiagen). For quantitative and semiquantitative PCR analysis, cDNA synthesis was performed using SuperScript III Reverse Transcriptase and Oligo (dT) primers (Invitrogen). Primers used for amplification are listed in Table S3.

For whole-genome expression analysis, Illumina Total Prep RNA amplification Kit (Ambion) was used according to manufacturer's guideline. Hybridization to Whole-Genome Expression BeadChips (HumanRef-8) was followed by analysis on an Illumina Beadstation 500. All samples were prepared in duplicates. Data analysis was conducted using manufacturer's Beadstudio software.

C-Peptide Release Assay.

C-peptide release was measured by incubating the cells in Krebs-Ringer solution containing bicarbonate and Hepes (KRBH; 129 mM NaCl/4.8 mM KCl/2.5 mM CaCl ₂/1.2 mM KH ₂PO ₄/1.2 mM MgSO ₄/5 mM NaHCO ₃/10 mM Hepes/0.1% BSA). The cells were incubated in KRBH buffer for 1 h to wash. The cells were incubated in KRBH buffer with 2.5 mM D-glucose for 1 h and then KRBH buffer with 20 mM D-glucose for 1 h. The C-peptide levels in culture supernatants were measured using the human C-peptide ELISA kit (Alpco Diagnostics).

Acknowledgments

We thank Kevin Eggan for organizational help and discussions, Anastasie Kweudjeu for help with transcriptional arrays, Danwei Huangfu for helpful discussions and experimental advice, Mariko Yamaki and Adriana Tajonar for technical help, Julian McKay-Wiggan, M.D. for performing skin biopsies, and Taylor Armstron and Sunanda Babu (University of Colorado, Aurora, CO) for islet antibody and HLA typing. S.C. is supported by the postdoctoral fellowship from Juvenile Diabetes Research Foundation. D.A.M. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the Harvard Stem Cell Institute (Kurtzig Fund), the Russell Berrie Foundation, the Handler Foundation, and the New York Stem Cell Foundation.

Footnotes

¹To whom correspondence should be addressed. E-mail: dmelton{at}harvard.edu

Author contributions: R.M., R.G., R.L.L., and D.A.M. designed research; R.M., S.C., M.S., and L.Y. performed research; T.L., R.G., and R.L.L. contributed new reagents/analytic tools; R.M., S.C., L.Y., R.L.L., and D.A.M. analyzed data; and R.M. and D.A.M. wrote the paper.

The authors declare no conflict of interest.

See Commentary on page 15523.

This article contains supporting information online at www.pnas.org/cgi/content/full/0906894106/DCSupplemental.

Freely available online through the PNAS open access option.

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Generation of pluripotent stem cells from patients with type 1 diabetes

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Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic β cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling.

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Leukaemia is a term used to describe many different kinds of cancers of the blood. However, in all of these diseases, too many white blood cells (also called leukocytes) are produced and these leukaemic cells do not mature normally. In the most serious types of leukaemia, these cells look like normal immature cells called blasts. But because they remain stuck in an immature state, these leukaemic blasts are not able to perform the functions of normal mature blood cells, which is to defend the body against infection and disease. In most forms of acute leukaemia, these leukaemic blasts accumulate in the bone marrow as well as in the blood and also suppress the formation of normal blood cells. The production of excessive numbers of cells in the blood and the lost ability to fight infection is what makes leukaemia rapidly fatal if not successfully treated.

Blood sample from a patient with acute leukaemia

Leukaemias are grouped according to the severity of the disease (how fast it is growing) and the types of white blood cells that become abnormal:

Acute leukaemias are diseases in which the leukaemic cells are rapidly growing and therefore need to be treated right away. They are usually sub-classified as acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) depending on the types of white blood cells that are affected.

Chronic leukaemias are diseases in which the leukaemic cells are growing more slowly and may still allow normal blood cell production to occur. Therefore, the symptoms may be much milder and the presence of the disease may escape diagnosis for several years before being detected. Chronic leukaemias are usually also sub-classified as either chronic lymphocytic leukaemia (CLL) or chronic myeloid leukaemia (CML) according to the types of white blood cells that are affected.

In addition, there are also other types and subtypes of leukaemia that have intermediate symptoms and pathology.

Both chronic and acute subtypes of leukaemia may develop in early childhood and then with increasing incidence as people get older. However, the properties of the leukaemic cells that arise in very young children are also somewhat different from those that arise in adults and their treatment is also more challenging.

Like most cancers, leukaemias are caused by a series of rare mutations (changes) in certain genes. These generally arise spontaneously at a very low rate inside primitive blood cell precursors throughout life. Very rarely, a mutated form of one of these genes may be inherited. When this happens the individual is highly predisposed to develop leukaemia. Other known, but also rare causes include accidental exposure to radiation or prior treatment with some types of anti-cancer drugs. However, in order for a full blown leukaemia to develop, it is thought that several changes usually have to be accumulated in order to permanently alter the programs that control normal blood cell production.

A large number of gene mutations have been linked to causation of human leukaemias, and in some cases, the same mutation is consistently and uniquely associated with a particular type of leukaemia. Chronic myeloid leukaemia (CML) is an example of a leukaemia where knowledge of a shared mutation led to the development of a drug (Gleevec) that is very successful in killing the leukemic cells in patients that are diagnosed with early stage (chronic phase) CML. However, in most patients with other types of leukaemia, the diversity of mutations that are present in their cells has made the development of effective "targeted" therapies more challenging.

Because many types of leukaemia are thought to require several rare mutations in order to develop, the first change is assumed to occur in a cell that will remain in the body for a long time. A prime candidate group of cells are the most primitive blood cell precursors - also called haematopoietic stem cells (HSCs). HSCs are responsible for making new blood cells in our bodies for our entire lives. If a stem cell is affected by a genetic change, all the cells it produces will inherit the same mutation. It has been shown that CML starts with a particular mutation in an HSC. However, the daughters of that first cell to be mutated then go through a number of steps to develop into mature white blood cells. The acquisition of new mutations can happen at any of these steps.

Healthy blood sample showing two white blood cells (neutrophils) and many red blood cells

'Blast crisis' in chronic myeloid leukaemia

Acute leukaemias usually require immediate and intensive treatment. Depending on the particular type of leukaemia and many other things about the individual patient, treatment options might include chemotherapy, steroids or a more intensive procedure involving high-dose chemotherapy followed by a transplant of healthy HSCs.

Two acute lymphocytic leukemia patients who are receiving chemotherapy; they are demonstrating some of the procedures used with chemotherapy

High-dose chemotherapy is the most effective currently established method to kill leukaemic cells and can cure some patients. However, it also severely damages the remaining normal blood-forming cells in the bone marrow. To replace these cells, patients are given a transplant that contains both HSCs as well as other important cells that contribute to the therapy. The cells for the transplant can be collected from the blood or bone marrow of a healthy donor. Such transplants are effective because they contain important immune cells that help to kill the leukaemic cells in addition to different subpopulations of HSCs that rescue blood production at different rates. A patient's own cells can sometimes also be used for the transplant, if it is possible to collect enough healthy cells before the treatment is performed. If a different donor is needed, they must match the patient's tissue type in order to reduce the risk of the transplanted donor cells being rejected.

Intensive chemotherapy followed by a HSC transplant continues to be one of the most effective ways of treating many types of acute leukaemia. However, the side effects of the procedure itself make it risky with the potential for early fatality and usually substantial after effects in long-term survivors, particularly in children. Therefore, this type of therapy is only considered when excellent matched donor cells are available and the features of the disease put it into a poor prognosis group.

Despite the successes of HSC transplant-based treatments of leukaemia, the use of cells from a normal donor can also have serious side effects:

Infections - The transplanted HSCs take time to produce the numbers of mature blood cells required to combat infections effectively, even when given "growth factors" that speed up this process. In the interval that thus inevitably precedes recovery, the patient is highly vulnerable to infections. Careful observation, restricted access to other people and preventative treatment with antibiotics are used to reduce infection risks.

Graft-versus-host disease (GvHD) - This complication occurs when donor blood cells attack the patient's own normal tissues. Symptoms include rashes, diarrhoea, blisters and fever. GvHD is a serious complication and can be life-threatening. It is minimized by closely matching the tissue type of the donor to the patient. This is easiest to achieve if the patient has a matched sibling. Other strategies to prevent GvHD include suppressing the immune system with drugs and removing a specific type of white blood cells (lymphocytes) from the initial transplant. However, it is now clear that the transplanted immune cells also play a critical role in eliminating the patient's leukemic cells. So a big challenge lies in obtaining a useful balance between achieving an effective graft-versus-leukemia (GvL) effect and a minimal GvHD effect.

Researchers and doctors are investigating ways to improve current transplantation approaches in order to address these limitations. Another challenge is the shortage of donors and several organisations are working to increase the number of volunteers in donor registries.

The high dose of chemotherapy given to leukaemia patients before a transplant is destructive to both the leukaemic cells and the patient's healthy bone marrow cells. Newer forms of transplantation called mini-allografts or reduced-intensity allografts have been developed to reduce the effects on the normal cells and boosting the GvL effect of the transplant. These procedures allow lower doses of chemotherapy to be used, which helps reduce the extent of damage to the bone marrow. Then, donor immune cells are transplanted with and after the donor HSCs to attack and eliminate any remaining leukaemic cells. This type of transplant has fewer side effects but some serious ones remain, particularly GvHD.

Several new techniques are now being tested in patients to see if they will prevent or reduce the severity of GvHD. Certain types of immune cells (regulatory T lymphocytes) that have a suppressive effect on the immune system may be administered to the patient. This can help prevent the donor immune cells from attacking the patient's own cells. Alternatively, attempts are being made to selectively remove the immune cells that cause GvHD from the initial transplant. Creating genetically engineered immune cells designed to specifically kill patients' leukaemic cells, particularly for certain lymphoid leukemias is also now showing great clinical promise.

Scientists and doctors are also currently investigating treatments that might reduce the time it takes for the patient's immune system to recover after a HSC transplant. Patients are now routinely given certain selected proteins called growth factors, which can enhance the production of cells needed to fight infections. This can help reduce the risk of infection while the patient recovers.

Another active area of research is the development of methods to expand HSCs in the laboratory. This strategy is of particular interest for enabling the use of cord blood cells collected and being frozen in a useful form from births taking place all over the world. These cells have very potent regenerative properties but are present in insufficient numbers to be widely useful in adults unless given as a combination of several collections.

Stem cell research is a rapidly developing field. New technologies such as the production of induced pluripotent stem cells (iPSCs) or producing HSCs by direct conversion of other cell types into HSCs are already being developed and used in the lab to study how normal human HSCs are maintained, and how human leukaemia arises. These studies are anticipated to provide new information about these cells, and thus how to design and test new, potentially more effective and less toxic therapies. Such research could revolutionize many of the current shortcomings of current transplants and lack of matched donors.

A bone marrow transplant is a stem cell therapy! The first one was carried out by E.D. Thomas in the late 1950s on a leukaemia patient using bone marrow from an identical twin.

Chronic myeloid leukaemia (CML) is a cancer of the granulocytes and is common in people who have been exposed to radiation. In this smear of peripheral blood from a patient with CML, the pink and purple cells are the white blood cells and the smaller, greyer cells are the red blood cells.

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Leukaemia is a term used to describe many different kinds of cancers of the blood. However, in all of these diseases, too many white blood cells (also called leukocytes) are produced and these leukaemic cells do not mature normally. In the most serious types of leukaemia, these cells look like normal immature cells called blasts.

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In most cases, Stargardt macular degeneration is caused by mutations in the gene. Less often, mutations in the gene cause this condition. The ABCA4 and ELOVL4 genes provide instructions for making proteins that are found in light-sensing (photoreceptor) cells in the retina.

The ABCA4 protein transports potentially toxic substances out of photoreceptor cells. These substances form after phototransduction, the process by which light entering the eye is converted into electrical signals that are transmitted to the brain. Mutations in the ABCA4 gene prevent the ABCA4 protein from removing toxic byproducts from photoreceptor cells. These toxic substances build up and form lipofuscin in the photoreceptor cells and the surrounding cells of the retina, eventually causing cell death. Loss of cells in the retina causes the progressive vision loss characteristic of Stargardt macular degeneration.

The ELOVL4 protein plays a role in making a group of fats called very long-chain fatty acids. The ELOVL4 protein is primarily active (expressed) in the retina, but is also expressed in the brain and skin. The function of very long-chain fatty acids within the retina is unknown. Mutations in the ELOVL4 gene lead to the formation of ELOVL4 protein clumps (aggregates) that build up and may interfere with retinal cell functions, ultimately leading to cell death.

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In most cases, Stargardt macular degeneration is caused by mutations in the gene. Less often, mutations in the gene cause this condition. The ABCA4 and ELOVL4 genes provide instructions for making proteins that are found in light-sensing (photoreceptor) cells in the retina. The ABCA4 protein transports potentially toxic substances out of photoreceptor cells.

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Leukemia is a cancer of the early blood-forming cells. Most often, leukemia is a cancer of the white blood cells, but some leukemias start in other blood cell types. There are several types of leukemia, which are divided based mainly on whether the leukemia is acute (fast growing) or chronic (slower growing), and whether it starts in myeloid cells or lymphoid cells. Different types of leukemia have different treatment options and outlooks.

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Leukemia is a cancer of the early blood-forming cells. Different types of leukemia have different treatment options and outlooks.

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A press release read, "Reprogramming a patient's own skin cells to replace cells in the brain that are progressively lost during Parkinson's disease has been shown to be technically feasible."

Many in the research community have found the results of this study were presented in a " clear and unbiased manner," and it appears to be a "solid scientific study." However, there are also articles debating various aspects of the study. I began to research this particular study, as well as clinical trials in general and stem cell research.

My editor suggested contacting the organizer of the study. Less than an hour later, Dr. Kwang-Soo Kim agreed to answer some questions that I emailed to him. I had the opportunity to be the voice of others with Parkinson's who may have the same questions.

So, here is my first experience as a journalist with Parkinson's.

Stem cells have more variety than I'd thought

Kim referred to this particular study as "very unusual and exceptional" because it was the first trial of its kind. It reported successfully using Lopez's own skin to create induced pluripotent stem cells, which are cells (usually skin cells) that are reprogrammed to behave like stem cells. Those cells, which are autologous, were then transplanted into the Parkinson's patient's brain. This is different from embryonic stem cells (ESC), which is an allogeneic approach.

OK, what do autologous and allogeneic mean? Allogeneic stem cells are not taken from one's own body, while autologous stem cells are created from one's own body.

Kim explained: "There are different approaches, such as fetal cells and embryonic stem cells. These approaches could be complementary and not exclusive. For instance, the ESC-based approach may be more economic and fast because the cells can be available for many patients. However, these cells are allogeneic and need immunosuppression. In contrast, autologous cell transplantation does not need immunosuppression ... but will take more time and budget. Once FDA approval is obtained, these different approaches can be compared with each other."

The pay-to-participate trial

Lopez paid $2 million to make the trial happen - he said this was both for his own benefit and to pave the way for others with Parkinson's. This has proven controversial.

"Medical and scientific groups, such as the International Society for Stem Cell Research, warn against 'pay to play' arrangements in which wealthy people buy their way into clinical trials," STAT's Sharon Begley noted.

However, Kim said that all pay-to-participate is under strict monitoring and regulation by the U.S. FDA and the Institutional Review Board.

He thinks personal financial status "does not/should not affect future studies and it is not a criteria in our next Phase I/II clinical trial, ... which can only handle a small number of patients."

As for future funding, they are seeking grants and philanthropic donations.

Kim also stated that "the most important issue is whether the patient fits to the criteria of our clinical team."

As a person who participates in clinical trials, hearing all of that was enough for me. My questions had been answered.

Paving the way to new horizons

Two years after the study began, time seems to be on Kim and Lopez's side. The cells are still alive, and the study has shown favorable outcomes. However, the one-person model was not a traditional blind study. Lopez knew he was receiving the therapy. Did his mindset create a placebo effect? Why only one patient?

"Since our approach is personalized therapy, even the big scale trial should start from an individual patient," Kim said. "So, we thought that we better start with a single patient and gain all experiences and information to move on."

The one-person study introducing personalized therapy seems to have paved the way for additional research. However, Kim noted that a double-blind study is needed to conclude its safety and efficacy.

I am grateful to Lopez who funded the trial and accepted the risks. He and Kim, together with all patients and doctors that participate in Parkinson's research, have the people with Parkinson's as their catalyst to keep pressing forward. Sometimes it's hard to see the forest through the trees, but the goal is the same: a therapy for us all. Ultimately, that is what is important - even if it is one person at a time.
*** Parkinson's News Today is strictly a news and information website about the disease. It does not provide medical advice, diagnosis or treatment . This content is not intended to be a substitute for professional medical advice, diagnosis, or treatment . Always seek the advice of your physician or another qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read on this website. The opinions expressed in this column are not those of Parkinson's News Today or its parent company, BioNews, and are intended to spark discussion about issues pertaining to Parkinson's disease.

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Taking a Closer Look at a First-ever Parkinson's Stem Cell Study

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Note: Parkinson's News Today is strictly a news and information website about the disease. It does not provide medical advice, diagnosis or treatment. This content is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Always seek the advice of your...

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Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk−/− but not Abca4−/− mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4−/− background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4−/− mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Journal Information

PNAS is the world's most-cited multidisciplinary scientific serial. It publishes high-impact research reports, commentaries, perspectives, reviews, colloquium papers, and actions of the Academy. In accordance with the guiding principles established by George Ellery Hale in 1914, PNAS publishes brief first announcements of Academy Members' and Foreign Associates' more important contributions to research and of work that appears to a Member to be of particular importance.

Publisher Information

The National Academy of Sciences (NAS) is a private, nonprofit organization of the country's leading researchers. The NAS recognizes and promotes outstanding science through election to membership; publication in its journal, PNAS; and its awards, programs, and special activities. Through the National Academies of Sciences, Engineering, and Medicine, the NAS provides objective, science-based advice on critical issues affecting the nation.

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Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration on JSTOR

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Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness.

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Type 1 diabetes (previously called insulin-dependent or juvenile diabetes) is usually diagnosed in children, teens, and young adults, but it can develop at any age.

If you have type 1 diabetes, your pancreas isn't making insulin or is making very little. Insulin is a hormone that enables blood sugar to enter the cells in your body where it can be used for energy. Without insulin, blood sugar can't get into cells and builds up in the bloodstream. High blood sugar is damaging to the body and causes many of the symptoms and complications of diabetes.

Type 1 diabetes is less common than type 2-approximately 5-10% of people with diabetes have type 1. Currently, no one knows how to prevent type 1 diabetes, but it can be managed by following your doctor's recommendations for living a healthy lifestyle, controlling your blood sugar, getting regular health checkups, and getting diabetes self-management education.

For Parents

If your child has type 1 diabetes, you'll be involved in diabetes care on a day-to-day basis, from serving healthy foods to giving insulin injections to watching for and treating hypoglycemia (low blood sugar; see below). You'll also need to stay in close contact with your child's health care team; they will help you understand the treatment plan and how to help your child stay healthy.

Much of the information that follows applies to children as well as adults, and you can also click here for comprehensive information about managing your child's type 1 diabetes.external icon

Causes

Type 1 diabetes is caused by an autoimmune reaction (the body attacks itself by mistake) that destroys the cells in the pancreas that make insulin, called beta cells. This process can go on for months or years before any symptoms appear.

Some people have certain genes (traits passed on from parent to child) that make them more likely to develop type 1 diabetes, though many won't go on to have type 1 diabetes even if they have the genes. Being exposed to a trigger in the environment, such as a virus, is also thought to play a part in developing type 1 diabetes. Diet and lifestyle habits don't cause type 1 diabetes.

Symptoms & Risk Factors

It can take months or years for enough beta cells to be destroyed before symptoms of type 1 diabetes are noticed. Type 1 diabetes symptoms can develop in just a few weeks or months. Once symptoms appear, they can be severe.

Some type 1 diabetes symptoms are similar to symptoms of other health conditions. Don't guess-if you think you could have type 1 diabetes, see your doctor right away to get your blood sugar tested. Untreated diabetes can lead to very serious-even fatal-health problems.

Risk factors for type 1 diabetes are not as clear as for prediabetes and type 2 diabetes, though family history is known to play a part.

Getting Tested

A simple blood test will let you know if you have diabetes. If you've gotten your blood sugar tested at a health fair or pharmacy, follow up at a clinic or doctor's office to make sure the results are accurate.

If your doctor thinks you have type 1 diabetes, your blood may also tested for autoantibodies (substances that indicate your body is attacking itself) that are often present with type 1 diabetes but not with type 2. You may have your urine tested for ketones (produced when your body burns fat for energy), which also indicate type 1 diabetes instead of type 2.

Management

Unlike many health conditions, diabetes is managed mostly by you, with support from your health care team (including your primary care doctor, foot doctor, dentist, eye doctor, registered dietitian nutritionist, diabetes educator, and pharmacist), family, teachers, and other important people in your life. Managing diabetes can be challenging, but everything you do to improve your health is worth it!

If you have type 1 diabetes, you'll need to take insulin shots (or wear an insulin pump) every day to manage your blood sugar levels and get the energy your body needs. Insulin can't be taken as a pill because the acid in your stomach would destroy it before it could get into your bloodstream. Your doctor will work with you to figure out the most effective type and dosage of insulin for you.

You'll also need to check your blood sugar regularly. Ask your doctor how often you should check it and what your target blood sugar levels should be. Keeping your blood sugar levels as close to target as possible will help you prevent or delay diabetes-related complications.

Stress is a part of life, but it can make managing diabetes harder, including controlling your blood sugar levels and dealing with daily diabetes care. Regular physical activity, getting enough sleep, and relaxation exercises can help. Talk to your doctor and diabetes educator about these and other ways you can manage stress.

Healthy lifestyle habits are really important, too:

Make regular appointments with your health care team to be sure you're on track with your treatment plan and to get help with new ideas and strategies if needed.

Whether you just got diagnosed with type 1 diabetes or have had it for some time, meeting with a diabetes educator is a great way to get support and guidance, including how to:

Develop and stick to a healthy eating and activity plan

Test your blood sugar and keep a record of the results

Recognize the signs of high or low blood sugar and what to do about it

Give yourself insulin by syringe, pen, or pump

Monitor your feet, skin, and eyes to catch problems early

Buy diabetes supplies and store them properly

Manage stress and deal with daily diabetes care

Ask your doctor about diabetes self-management education and to recommend a diabetes educator. You can also search the American Association of Diabetes Educators' nationwide directoryexternal icon for a list of educators in your community.

Hypoglycemia

Hypoglycemia pdf icon [PDF - 100KB] (low blood sugar) can happen quickly and needs to be treatedexternal icon immediately. It's most often caused by too much insulin, waiting too long for a meal or snack, not eating enough, or getting extra physical activity. Hypoglycemia symptoms are different from person to person; make sure you know your specific symptoms, which could include:

Shakiness

Nervousness or anxiety

Sweating, chills, or clamminess

Irritability or impatience

Dizziness and difficulty concentrating

Hunger or nausea

Blurred vision

Weakness or fatigue

Anger, stubbornness, or sadness

If you have hypoglycemia several times a week, talk to your doctor to see if your treatment needs to be adjusted.

Connect with Others

Tap into online diabetes communities for encouragement, insights, and support. The American Diabetes Association's Community pageexternal icon and the American Association of Diabetes Educators' Peer Support Resourcesexternal icon are great ways to connect with others who share your experience.

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Type 1 diabetes (previously called insulin-dependent or juvenile diabetes) is usually diagnosed in children, teens, and young adults, but it can develop at any age. If you have type 1 diabetes, your pancreas isn't making insulin or is making very little.

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Abstract

Malignant stem cells have recently been described as the source of several types of human cancer. These unique cell types are typically rare and possess properties that are distinct from most other tumor cells. The properties of leukemic stem cells indicate that current chemotherapy drugs will not be effective. The use of current cytotoxic agents is not effective in leukemia because the agents target both the leukemic and normal stem cell populations. Consequently, new strategies are required that specifically and preferentially target the malignant stem cell population, while sparing normal stem cells. Several well known agents are lethal for the leukemic stem cell in preclinical testing. They include parthenolide, commonly known as feverfew, and TDZD-8. They have undergone various levels of preclinical development, but have not been used in patients as yet in the cancer setting. These drugs and combinations of existing therapies that target the leukemic stem cell population may provide a cure in this disease. This article summarizes recent findings in the leukemic stem cell field and discusses new directions for therapy.

Keywords: leukemic stem cell, parthenolide, TDZD-8

Introduction

Approximately 50 years ago the concept was introduced that a stem cell may be connected to the origin and evolution of myeloid leukemia. To hypothesize the evolution of this disease, some form of normal stem or progenitor cell undergoes a mutation, giving rise to an entity that is functionally defined as a leukemic stem cell. The normal stem cells continue to differentiate into the hematopoietic lineage giving rise to erythrocytes, platelets, leukocytes, and granulocytes. The mutated stem cells have properties similar to the normal stem cells and can also differentiate into the hematopoietic lineage carrying the defect/s or can remain and accumulate as immature progenitor cells, also known as blast cells ().

The chemotherapeutic agents used today effectively eradicate the blast cells in many patients. However, those same agents have very little if any activity at the level of the blast progenitor cell, the leukemic stem cell (LSC), which is biologically distinct from most of the cells that are found in a typical patient. An LSC is a functionally defined entity not necessarily named because it arises from a normal stem cell, but because it fulfills the same criteria used to define normal stem cells. These cells can undergo self renewal, are multipotent, and highly proliferative. The origin of the LSC has been the subject of considerable research in recent years.

During normal developmental progression from stem cell to progenitor cell to mature cell, mutations may potentially occur at any point during this evolution, giving rise to a malignant entity. A mutation in a normal stem cell might give rise to a unit that could be considered an LSC. However, there is experimental evidence that suggests that mutations in a progenitor cell that no longer has all the characteristics of a stem cell can also give rise to an entity that can initiate and maintain leukemic disease. By extrapolation, it can be seen that more differentiated cells with the appropriate mutations may also give rise to the leukemic stem cell.

From a therapeutic perspective, the nature of the LSC may vary depending upon the stage during which it arose. Accordingly, drug resistance and various characteristics that are relevant to therapy may also differ, based on the origin of the diseased cell. Thus, it is possible to speculate that certain forms of leukemia that are relatively amenable to current therapies may derive from more differentiated cells that have certain intrinsic properties that are more readily addressed by conventional therapy. A plethora of new agents is available for treating leukemias, including kinase inhibitors, histone deacetylase inhibitors, cyclin D kinase inhibitors, heat shock protein inhibitors, methylation inhibitors, farnesyltransferase inhibitors, NF-kB inhibitors, and proteasome inhibitors., All of these agents affect specific mechanisms that may have gone awry. Despite the excitement and potential of all of these agents, there is not a single drug that has yet been validated as useful for eradicating the human LSC. An extremely important undertaking will be to fill this gap and to understand if and how all these new therapies are acting at the level of the stem cell.

Because anthracyclines, alkylating agents, nucleoside analogs, and topoisomerase inhibitors currently used in the treatment of acute myeloid leukemia (AML) often fail, they may not be targeting LSCs very effectively. In fact, there is no evidence that there is any selectivity or specific targeting of a leukemic versus normal stem cell by these agents. For example, Ara-C, which is a cycle-active agent commonly used in treating leukemia, shows virtually no activity with isolated LSCs. In contrast, this agent is effective when tested on blast cells from the same patient. There is a distinct difference in treating these two cell populations. When anthracyclines are tested in vitro, they are extremely cytotoxic both to LSCs and normal stem cells and there is no selectivity. However, there are ways that conventional therapies can be used in the appropriate combinations and appropriate methodologies, some of which have been previously published, to enhance the selective targeting and killing of leukemic stem cells.

The leukemic stem cell

LSCs can be isolated based on their cell surface markers using the currently available cell-sorting technologies. Once the leukemic stem cells have been isolated, they can be analyzed using the same techniques applied in any type of cancer cell to understand the specific mutations and pathways that propel growth and survival. Tumor-associated properties in the LSC could include mutations in the kinase domains, transcription factors, and tumor suppressors, or alterations in the growth and survival mechanisms mediated through NF-kappa B (NF-κB) or PI3 kinase, or changes in physiology, glucose metabolism, or responses to oxidative stress, to name a few. The results obtained from these analyses of LSCs can be compared to the unique properties of normal stem cells, which are relatively rare, have a quiescent cell cycle status, can efflux drug, and have self-renewal properties. Based on these findings we will be able to effectively develop regimens that will target LSCs.

From this relatively broad perspective, it is difficult to choose the most effective pathways to target. A great deal of knowledge has been amassed on agents that target mitogenic tyrosine kinase mutations, such as the BCR/ABL mutation by imatinib mesylate, which induces apoptosis of primary chronic myeloid leukemia (CML) cells. Another example is the Flt3 inhibitor CEP701, which induces apoptosis of primary AML blasts. However, there is no evidence that inhibiting these pathways is relevant to inhibiting the proliferating LSC.

As an alternative to inhibiting the activity of specific mutations in a cancer cell, it might be appropriate to consider the physiology of other unique mechanisms that maintain the viability and survival of these cells. Two such mechanisms have been suggested in the literature: the constitutive activation of the PI3 kinase pathway and NF-κB, which are evident in LSCs. Inhibiting these two pathways might have therapeutic relevance. While no particular mutations or specific genetic events are associated with activation of the NF-κB or PI3-kinase pathways, converging events, such as multiple different mutations, may feed into these pathways. However, such pathway modification may not be the only mechanism to produce leukemia.

Primitive human LSC populations can be selected by cell surface markers containing CD34+/CD38−/CD123+ antigens. These cells are almost entirely quiescent, mimicking normal stem cells. As a result, cell cycle agents that are active in dividing cells will not be effective with this population. To characterize unique properties of LSCs, these purified populations have been examined using molecular analyses that look for activation of the NF-κB pathway. As shown in , activation of the NF-κB pathway can be readily detected. Normal hematopoietic stem cells do not show activation of NF-κB. We believe that this is a leukemia-specific phenomenon.

New agents to target the leukemic stem cell

Idarubicin with a proteasome inhibitor, parthenolide, and TDZD-8 are three examples of regimens that selectively target the leukemic stem cell. All three are able to inhibit NF-κB activity. The combination of idarubicin with various classes of proteasome inhibitors mediates selective apoptosis in LSCs while sparing normal cells. The second agent, parthenolide, is a naturally occurring sesquiterpene lactone that also selectively kills LSC populations with minimal activity in normal stem cells. Continuing studies with this agent in colony-forming assays have been used to measure progenitor cell function. This pattern of selectivity continues with the third agent, TDZD-8, which is lethal only to the leukemic population. Data on TDZD-8 is yet unpublished. From a preclinical standpoint, these types of molecules appear promising.

Parthenolide

Parthenolide is a small molecule that is the active compound in the plant known as feverfew, which has been used for centuries in herbal remedies to treat headache and inflammation. It is a potent inhibitor of NF-κB. Parthenolide selectively ablates myeloid leukemia cells at an IC ₅₀ of 2.5 μM and causes apoptosis in primary human AML cells and blast crisis CML (bcCML) cells. AML progenitor and stem cells were analyzed in in vitro colony forming assays in a nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft mouse model. Parthenolide preferentially targeted both AML progenitor and LSC populations. The only drawback is that parthenolide is not a good candidate for pharmacologic development because it is not particularly water soluble. We have collaborated with Peter A. Crooks, PhD, at the University of Kentucky to develop analogs of parthenolide. The best candidate so far is a fumarate salt of dimethylaminoparthenolide. Its solubility in water is ~1000-fold greater than parthenolide and maintains its LSC-specific activity. It has been picked up by the NCI's RAID program and is currently undergoing further preclinical development. We hope to see this agent entering trials relatively soon.

TDZD-8

TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) belongs to a family of molecules with an interesting history. A research group from the University of Madrid tested agents to inhibit the enzymatic activity of glycogen synthase kinase-3 beta (GSK-3β with the aim of treating neurologic diseases, such as Alzheimer's, in which the enzyme GSK-3β is very active. The agents they tested also have NF-κB inhibitory activity. One of these compounds, TDZD-8, is a non-ATP competitive inhibitor of GSK-3β (IC ₅₀=2μM), binds to the active site of GSK-3β, and inhibits the activities of Cdk1/cyclin B, casein kinase-II, and protein kinase A and C (IC ₅₀>100μM). Preliminary data show that TDZD-8, similar to parthenolide, selectively induces the death of primary AML progenitor cells.

Further preliminary studies compared the efficacy of treating the AML progenitor cells with either parthenolide or TDZD-8. An overnight culture of AML progenitor cells expressing CD34+CD38- treated with either parthenolide or TDZD-8 showed that survival is rapidly impaired by TDZD-8 in comparison to parthenolide (half life of 120 minutes for cells in the presence of TDZD-8 vs > 360 minutes for those in the presence of parthenolide), relative to untreated controls. By contrast, there was very little to no activity in normal patient specimens.

In terms of the kinetics of these two agents, cells were treated with TDZD-8 or parthenolide at intervals of 30, 60, 120 ...1440 minutes and then assayed for viability. It became readily apparent between 6 and 12 hours that cells were dying in the presence of parthenolide. With TDZD-8, these cells were killed in as little as 30 minutes. When AML progenitor cells were introduced into NOD/SCID mice to rigorously assess stem cell potential, TDZD-8 inhibited the engraftment of AML leukemic stem cells, but did not significantly inhibit engraftment of normal hematopoietic stem cells. In a phenotypic analysis, cells were taken from the cell viability assay and introduced at each of the same time points into a functional assay such as progenitor cell colony-forming assays with striking results. The cells from the previous assay that were transferred into a functional assay had no detectable colony forming activity in as little as 30 minutes in the presence of TDZD-8. By comparison, parthenolide required an overnight time lapse before readily evident cell kill was observable. The mechanism of TDZD-8 is being investigated; however, from an empirical perspective, the extremely fast cell death with this agent is a very exciting development.

Conclusion

The two different agents discussed in this paper, parthenolide and TDZD-8, are chemically distinct entities. These compounds have the property of mediating cell death and inhibiting leukemic stem cell-specific activity. The empirical observation is that there are two types of stimuli required to bring about leukemia-specific cell death. The first is the inhibition of a survival pathway. The NF-kappa B and PI3 kinase pathways are affected by these drugs, but there could be others. Yet inhibiting pathways alone is not particularly toxic. However, when combined with a stimulus in the form of oxidative and/or genotoxic stress, the two signals together appear to be highly selective in killing leukemic stem cells, but are not particularly toxic at the normal stem cell level. Agents like parthenolide and TDZD-8 appear to simultaneously deliver both signals (). A general criterion for selecting therapeutic regimens could be the ability of the agent to deliver both signals simultaneously.

In developing regimens that are more selective to leukemic stem cells, these agents should be developed for patient use and their activity should be validated at the level of stem cells. Similarly, when these regimens are used in patients, patient outcomes should be assessed in terms of the relative efficacy of targeting leukemic stem cells in vivo. The assays are currently available and can be applied to determine this.

Footnotes

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References

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Malignant stem cells have recently been described as the source of several types of human cancer. These unique cell types are typically rare and possess properties that are distinct from most other tumor cells. The properties of leukemic stem cells indicate ...

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Harvard Stem Cell Institute collaborations are revealing new drug targets for Parkinson's and ways to restore brain cells damaged by the disease.

What do Muhammad Ali and Michael J. Fox have in common? They are both extraordinary individuals who developed an all-too-common disease and developed it at a particularly tragic early age.

Approximately one to two percent of the population over the age of sixty receives a diagnosis of Parkinson's disease, although scientists believe that may be a very conservative estimate. At least one million Americans are currently known to be living with the condition, which begins with a slight tremor of the hands and progresses until the patient cannot walk without assistance.

These symptoms are associated with the death of cells in a part of the brain called the substantia nigra, which produces the neurotransmitter dopamine. The cause of the cell death that begins the symptoms of Parkinson's is unknown, although a combination of genetic and environmental factors is suspected.

New Possibilities

Harvard Stem Cell Institute (HSCI) researchers are making rapid progress in our understanding of Parkinson's disease.

A Patient Disease Model

HSCI Principal Faculty member and Director of the Neuroregeneration Research Institute at McLean Hospital Ole Isacson, MD, with funding from HSCI, the Harvard Miller Consortium for the Development of Nervous System Therapies, and the National Institutes of Health, has generated brain cells that produce dopamine, collected from the skin cells of patients with Parkinson's.

Isacson orchestrated the transformation by biologically reprogramming the mature skin cells into induced pluripotent stem cells, and then encouraging the stem cells to become dopaminergic neurons. Neurons were also made from skin cells collected from individuals with genetic mutations associated with high risk for Parkinson's disease.

The creation of this in vitro disease model provides a powerful platform for studying Parkinson's outside of the body. In the journal Science Translational Medicine, Isacson and his team reported that many of the mutations implicated in Parkinson's affect the function of the mitochondria, the cellular organelle responsible for energy production. In collaboration with HSCI Director of Translational Medicine Lee Rubin, PhD, Isacson's lab started to identify compounds that could eliminate disease symptoms in cell lines derived from people carrying Parkinson's mutations.

Repairing the Brain

HSCI scientists are also exploring how to replace the neurons destroyed in Parkinson's disease and other nervous system disorders. The lab of HSCI Principal Faculty member Paola Arlotta, PhD, is developing new approaches to restore nervous system function by turning one type of already differentiated motor neuron into another. Her work, which is primarily used to study Amyotrophic Lateral Sclerosis (commonly referred to as ALS or Lou Gehrig's disease), has broad implications for Parkinson's. Arlotta wants to know how these reprogrammed cells function within the brain and use this understanding to recreate brain cell connections destroyed by ALS and Parkinson's.

The lab of HSCI Principal Faculty member Vik Khuana is tackling Parkinson's and other neurodegenerative diseases that are caused when neurons are gummed up by abnormally folded and clumped up proteins. Revolutionary advances in stem cell technology are enabling his lab to take a small piece of skin from a patient and convert the skin cells into stem cells (so-called "induced pluripotent stem cells" or iPS cells). From these stem cells, Khurana's team can make any tissue in the body, including brain cells. So, for the first time in history, scientists can observe the abnormalities of these diseases develop "in a dish." In 2013, his lab identified abnormalities that result from protein misfolding in stem cell-derived neurons from Parkinson's disease patients. More importantly, they showed that such abnormalities could be reversed by manipulating patient genes ("genome editing") or exposing the cells to drug-like molecules.

Another facet of Parkinson's disease is being pursued by HSCI Affiliate Faculty member U. Shivraj Sohur, MD, PhD. He and his colleagues focus on the molecular changes that cause the growth of nerve cells in both development and adulthood. These studies are the first steps to repairing brain regions affected by diseases like Parkinson's.

HSCI investigators are taking a range of complementary approaches that will aid our understanding of what causes Parkinson's disease, how it progresses, and how damaged neurons can be replaced. The collaborations made possible by the HSCI network will accelerate the translation of basic Parkinson's research into clinical treatments for the disease.

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Harvard Stem Cell Institute collaborations are revealing new drug targets for Parkinson's and ways to restore brain cells damaged by the disease. What do Muhammad Ali and Michael J. Fox have in common? They are both extraordinary individuals who developed an all-too-common disease and developed it at a particularly tragic early age.

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Promises and Dangers of Stem Cell Therapies | Daniel Kota | TEDxBrookings