Stem cell transplantation helps patients with diabetes become insulin free

The majority of patients with type 1 diabetes who underwent a certain type of stem cell transplantation became insulin free, several for more than three years, with good glycemic control, and also increased C-peptide levels, an indirect measure of beta-cell function, according to a study in the April 15 issue of JAMA, a theme issue on diabetes.

Richard K. Burt, M.D., of the Northwestern University Feinberg School of Medicine, Chicago, presented the findings of the study at a JAMA media briefing at the National Press Club in Washington, D.C.

Clinical evidence indicates that there is an inverse association between beta-cell (a type of cell in the pancreas that secretes insulin) preservation and function and chronic complications of type 1 diabetes mellitus (DM), and the higher the C-peptide levels (a byproduct of insulin production, made up of amino acids), the lower the incidence of some types of complications of type 1 DM. A previous study found that autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with newly diagnosed type 1 DM resulted in the majority of patients becoming insulin free during the follow-up, which averaged about 19 months. "However, it was suggested that subsequent insulin independence was a prolonged honeymoon period due to dietary and exercise changes associated with close posttransplant medical observation," the authors write, and it was not known if this change was because of an improvement in beta-cell preservation.

HSCT, which uses a patient's own blood stem cells, involves the removal and treatment of the stem cells, and their return to the patient by intravenous injection.

Dr. Burt and colleagues conducted a study to determine if posttransplant insulin independence was due to improved beta-cell function by monitoring the C-peptide levels of 23 patients who underwent stem cell transplantation. The patients, with type 1 DM, were ages 13-31 years.

Of the 23 patients, 20 experienced time free from insulin (12 continuously and 8 transiently). Patients remained continuously insulin free for an average time of 31 months (range, 14-52 months). One patient had more than 4 years with no exogenous (produced outside the body) insulin use, 4 patients for at least 3 years, 3 patients for at least 2 years, and 4 patients for at least 1 year. Eight patients relapsed and resumed insulin use at low doses. The majority of patients achieved good glycemic control.

In the continuously insulin-free group, average area under the curve (AUC; a type of measurement) of C-peptide levels before transplantation (225.0 ng/mL per 2 hours) showed a significant increase at 24 months after transplantation (785.4 ng/mL per 2 hours) and at 36 months after transplantation (728.1 ng/mL per 2 hours). In the transient insulin–independent group, average AUC of C-peptide levels also increased from 148.9 ng/mL per 2 hours pretransplantation to 546.8 ng/mL per 2 hours at 36 months, which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with the antihyperglycemic drug sitagliptin, which was associated with an increase in C-peptide levels.

Two patients developed pneumonia in the hospital, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia (sperm deficiency). There were no deaths.

"In conclusion, autologous nonmyeloablative HSCT was able to induce prolonged and significant increases of C-peptide levels associated with absence of or reduction of daily insulin doses in a small group of patients with type 1 DM," the researchers write. "At the present time, autologous nonmyeloablative HSCT remains the only treatment capable of reversing type 1 DM in humans. Randomized controlled trials and further biological studies are necessary to confirm the role of this treatment in changing the natural history of type 1 DM."

K-State engineers create DNA sensors that could identify cancer using material only one atom thick

Kansas State University engineers think the possibilities are deep for a very thin material.

Vikas Berry, assistant professor of chemical engineering, is leading research combining biological materials with graphene, a recently developed carbon material that is only a single atom thick.

"The biological interfacing of graphene is taking this material to the next level," Berry said. "Discovered only four years ago, this material has already shown a large number of capabilities. K-Staters are the first to do bio-integrated research with graphene."

To study graphene, researchers rely on an atomic force microscope to help them observe and manipulate these single atom thick carbon sheets.

"It's a fascinating material to work with," Berry said. "The most significant feature of graphene is that the electrons can travel without interruptions at speeds close to that of light at room temperature. Usually you have to go near zero Kelvin -- that's about 450 degrees below zero Fahrenheit -- to get electrons to move at ultra high speeds."

One of Berry's developments is a graphene-based DNA sensor. When electrons flow on the graphene, they change speed if they encounter DNA. The researchers notice this change by measuring the electrical conductivity. The work was published in Nano-Letters.

"Most DNA sensors are optical, but this one is electrical," Berry said. "We are currently collaborating with researchers from Harvard Medical School to sense cancer cells in blood."

Another area he is exploring is loading graphene with antibodies and flowing bacteria across the surface.

"Most researchers focus on pristine graphene, but we're making it dirty," he said.

Berry and Nihar Mohanty, a graduate student in chemical engineering, used a type of bacteria commonly found in rice and interfaced it with graphene. They found that the graphene with tethered antibodies will wrap itself around an individual bacterium, which remains alive for 12 hours.

Berry said that possible applications include a high-efficiency bacteria-operated battery, where by using geobater, a type of bacteria known to produce electrons, can be wrapped with graphene to produce electricity. The research was presented at the annual American Physical Society conference in Pittsburgh and the American Institute for Chemical Engineers conference in Philadelphia.

"Materials science is an incredible field with several exploitable quantum effects occurring at molecular scale, and biology is a remarkable field with a variety of specific biochemical mechanisms," Berry said. "But for the most part the two fields are isolated. If you join these two fields, the possibilities are going to be immense. For example, one can think of a bacterium as a machine with molecular scale components and one can exploit the functioning of those components in a material device."

For his doctoral research, Berry used bacteria to make a humidity sensor.

"That was only possible through combining materials science with biological science," he said.

Another area of his current research is compressing and stretching molecular-junctions between nanoparticles. Berry said that his group has developed a molecular-spring device where they can compress and stretch molecules, which then act like springs, allowing researchers to study how they relax back. He said that this technology could be used to create molecular-timers in which the spring action from a decompressed molecule on a chip could trigger a circuit, for instance.

Berry said for stretching the molecules, Kabeer Jasuja, a doctoral student in chemical engineering, came up with the idea to place the device on a centrifuge to stretch the molecules with centrifugal force.

The work was published in the journal Small.

Creating ideal neural cells for clinical use

A JOLLA, Calif., April 13, 2009 -- Investigators at the Burnham Institute for Medical Research (Burnham) have developed a protocol to rapidly differentiate human embryonic stem cells (hESCs) into neural progenitor cells that may be ideal for transplantation. The research, conducted by Alexei Terskikh, Ph.D., and colleagues, outlines a method to create these committed neural precursor cells (C-NPCs) that is replicable, does not produce mutations in the cells and could be useful for clinical applications. The research was published on March 13 in the journal Cell Death and Differentiation.

When the C-NPCs created using the Terskikh protocol were transplanted into mice, they became active neurons and integrated into the cortex and olfactory bulb. The transplanted cells did not generate tumor outgrowth.

"The uniform conversion of embryonic stem cells into neural progenitors is the first step in the development of cell-based therapies for neurodegenerative disorders or spinal injuries," said Dr. Terskikh. "Many of the methods used to generate neural precursor cells for research in the lab would never work in therapeutic applications. This protocol is very well suited for clinical application because it is robust, controllable and reproducible."

Dr. Terskikh notes that the extensive passaging (moving cells from plate to plate) required by some protocols to expand the numbers of neural precursor cells limits the plasticity of the cells, can introduce mutations and may lead to the expression of oncogenes. The Terskikh protocol avoids this by using efficient conversion of hESCs into primary neuroepithelial cells without the extensive passaging.

The scientists were able to rapidly neuralize the hESCs by culturing them in small clusters in a liquid suspension. The cells developed the characteristic "rosettes" seen in neuroepithelial cells. The C-NPCs were then cultured in monolayers. Immunochemical and RT-PCR analysis of the cells demonstrated that they were uniformly C-NPCs. Whole-genome analysis confirmed this finding. Immunostaining and imaging showed that the cells could be differentiated into three distinct types of neural cells. The team then demonstrated that the C-NPCs differentiated into neurons after transplantation into the brains of neonatal mice.

This research received funding from the National Institutes of Health and the California Institute for Regenerative Medicine.

When cancer cells can't let go

Like a climber scaling a rock face, a migrating cancer cell has to keep a tight grip on the surface but also let go at the right moment to move ahead. Chan et al. reveal that the focal adhesion kinase (FAK) coordinates these processes to permit forward movement. The study will be published online April 13 (http://www.jcb.org/) and will appear in the April 20 print issue of the Journal of Cell Biology.
Crawling cancer cells send out extensions called invadopodia. By releasing enzymes that dissolve the extracellular matrix (ECM), invadopodia clear a path for the cell to wriggle through. As they move, cancer cells get traction by temporarily attaching to the ECM through focal adhesions. FAK spurs focal adhesions to disengage, and it is more abundant in metastatic tumors. Whether FAK also regulates invadopodia was unknown.
When Chan et al. removed FAK, breast cancer cells were much less invasive. But to the team's surprise, the FAK-lacking cells sprouted extra invadopodia. The cells also sported large focal adhesions that were particularly sticky. The protein Src serves as FAK's helper. FAK and Src work together to phosphorylate tyrosines in proteins such as paxillin, which then disassemble the focal adhesion. But the team found that in cells missing FAK, the phosphorylated proteins accumulated in invadopodia. Src's localization reflects this difference. In control cells, Src accumulated in focal adhesions. In FAK's absence, Src headed to the invadopodia.
The work suggests that FAK controls movement by balancing the number of invadopodia that create a path for migration and the number of focal adhesions that hold the cell back. The next question, the researchers say, is how FAK and Src integrate these events to promote invasion.

UCSF team closer to creating safe embryonic-like stem cells

A team of UCSF researchers has for the first time used tiny molecules called microRNAs to help turn adult mouse cells back to their embryonic state. These reprogrammed cells are pluripotent, meaning that, like embryonic stem cells, they have the capacity to become any cell type in the body.
The findings suggest that scientists will soon be able to replace retroviruses and even genes currently used in laboratory experiments to induce pluripotency in adult cells. This would make potential stem cell-based therapies safer by eliminating the risks posed to humans by these DNA-based methods, including alteration of the genome and risk of cancer.
"Using small molecules such as microRNAs to manipulate cells will play a major role in the future of stem cell biology," says senior author Robert Blelloch, MD, PhD, of the Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research at UCSF.
Scientists are interested in reprogramming because it would offer a way to create cells that provide a genetic match for individual patients. A patient's skin cells could be reverted to pluripotent cells in the culture dish and then prompted to differentiate into adult cells, such as those of the heart, lung and brain. These cells could then be transplanted into patients, without the fear of rejection.
The study, reported in the April 12, 2009 advanced online edition of the journal "Nature Biotechnology" and scheduled for the May 8, 2009 print issue, used a combination of microRNAs and retrovirus-introduced genes to transform fibroblast cells -- found throughout the body of mice and humans -- into pluripotent cells.
The current finding comes on the heels of a study published by the group in the December 2008 print edition of "Nature Genetics" that showed that microRNAs, which can be synthesized in the lab, encouraged embryonic stem cells to self-replicate, a finding that has implications for replicating stem cells in the culture dish and exploring stem cells' role in cancers.
Previous methods for creating embryonic stem cell-like cells have relied on the introduction of DNA that encodes four transcription factors, proteins that play a role in the production of genes. The limitation of this method is that three of the four genes that code for these transcription factors -- oct4, klf4 and c-myc – are oncogenes, meaning they promote the uncontrolled cell growth characteristic of cancer.
In the current study, led by Robert Judson, a graduate student in the Blelloch lab, the scientists induced pluripotency using a combination of infection and transfection. The infection involved introducing three viruses, each containing a transcription factor known to induce pluripotency. The transcription factor for c-myc was not included. The transfection involved a simple process in which the tiny microRNA molecules were mixed with a lipid, allowing them to pass through the cell membrane. By labeling the fibroblast cells, they showed that the treated cells could be incorporated into a mouse embryo and become every cell type in the adult animal -- including germline cells that would produce the next generation of mice.
"These are transient, non-coding molecules that do not incorporate into the genome, but promote self-replication and have the potential to induce pluripotency," Blelloch says. "They do their thing -- turn a somatic cell into an embryonic stem cell-like one -- and then they're gone."
"MicroRNAs give us a new tool to manipulate the fate of cells," Blelloch says.
MicroRNAs are snippets of single-stranded RNA that prevent a gene's code from being translated from messenger RNA into protein. They were debuted in 1993, when scientists reported the discovery of a microRNA in the microscopic roundworm C. elegans. Since then, the field has "exploded," says Blelloch, with hundreds of microRNAs discovered in the last eight years across a broad range of species, from plants to animals.
Produced in the nucleus and released into the cytoplasm, microRNAs home in on messenger RNAs that share part of their genetic sequence. When they find them, they latch on, preventing the messenger RNA from being processed by the protein-making machines known as ribosomes. As such, microRNAs are able to ratchet down a cell's production of a given protein.
Currently, Blelloch and his colleagues are working to replace all four transcription factors with microRNAs and conducting experiments that will reveal the mechanism by which these small molecules are able to induce pluripotency. The team will also be looking to determine which microRNAs might be able to turn adult cells directly into particular adult cell types, by-passing the embryonic stem cell-like stage altogether.
"The goal now is to ensure the safety of induced pluripotent stem cells and to differentiate them into cells that can be used to repair damaged tissue and treat disease," he says.

Dental assessment prior to stem cell transplant: treatment need and barriers to care

Objective To assess the treatment needs of patients undergoing pre-haematopoietic stem cell transplant (HSCT) dental assessment, to collate the examination findings and treatment provided and to define the management issues impacting on care.Design Single centre retrospective analysis.Setting Salaried Primary Care Dental Service, Western General Hospital, Edinburgh, UK.Subjects and methods One hundred and sixteen available charts of patients who attended for pre-transplant dental assessment during April 2004-June 2007 were examined.Results Ninety-four patients, 52 men (55.3%) and 42 women (43.6%), were included. Patients were referred a mean of 31.5 (SD 18.82) days before admission for transplant. Dental assessment occurred, on average, 7.88 days (SD 6.78) following referral. Eighty-eight (93.6%) patients were dentate, while six (6.3%) were edentulous. Eighty-eight (93.6%) patients presented with oral disease; 89 (94.7%) patients received dental care. Issues impacting on care were medical (n = 88, 93.6%), time constraints (n = 73, 77.7%), no GDP (n = 25, 26.7%), dental complexity (n = 5, 5.3%) and anxiety management (n = 1, 1.1%).Conclusion The majority of patients required dental care, most of which, for healthy adults, would normally be completed within a primary care setting. However, the issues surrounding the care of patients destined for HSCT indicate that there is a place for a dedicated dental service as part of the multidisciplinary team.

Durey K, Patterson H, Gordon K.

GPT, Edinburgh Postgraduate Dental Institute, Floor 4, Lauriston Building, Lauriston Place, Edinburgh, EH3 9HA.

Tumor initiating cancer stem cells from human breast cancer cell lines.

Breast cancer is composed of heterogeneous cell populations with different biological properties. The capacity to form tumors resides in a small group of cells termed tumor initiating cells or cancer stem cells. Tumor initiating cells have been identified in a variety of cancers by sorting of subpopulations based on cell surface markers and transplantation into animal models. Tumor initiating cells have the important feature of self renewal, which is a property in common with stem cells. We examined established breast cancer lines for cells with tumor initiating properties. A dye efflux side population in MCF7 and T47D lines expressed markers of breast cancer stem cells. The side population represents a distinct morphologic and functional subpopulation within the human breast cancer cell lines MCF7 and T47D. The side population from human breast cancer cell lines was able to initiate tumors in vivo. The side population cells from human breast cancer cell lines were more resistant to ionizing radiation than the non-side population. We concluded that tumor initiating cells exist in established human breast cancer cell lines.

Han JS, Crowe DL.

University of Illinois Cancer Center, Chicago, IL 60612, USA.