FUNCTIONAL MAGNETIC RESONANCE IMAGING CHARACTERIZATION OF COGNITIVE TASKS IN PATIENTS WITH FRAGILE X-ASSOCIATED TREMOR ATAXIA SYNDROME

PSTP Student:  Teresa Barcellos
Summer Mentor:  Dr. Susan Rivera
Collaborators:  David N, and Henry M

Fragile X is an X-linked genetic disorder characterized by mental retardation, social deficits, such as social anxiety and autism, and physical findings, including large, prominent ears, macroorchidism, hyperextensible joints, flat feet, and mitral valve prolapse.  The disorder is caused by a trinucleotide expansion in the 5’ untranslated region (UTR) of the FMR1 gene.  Normal individuals have fewer than 55 CGG repeats in the UTR, while patients with Fragile X have more than 200 repeats.  Individuals with repeat numbers between 40 and 240 are considered premutation carriers of the disorder.  In persons with the full mutation, the gene is hypermethylated and consequently is rarely translated.  As a result, these individuals have no or low levels of the gene product, FMRP.  Clinically, severity of the Fragile X phenotype shows positive correlation with serum level of FMRP.  Historically the premutation has not been associated with a particular phenotype, although female carriers have a higher incidence of premature ovarian failure (Hagerman et al, 2001).  Additionally, some studies have shown an increased incidence of social anxiety disorders among carriers (Hagerman and Hagerman, 2002).  Interestingly, carriers with social anxiety or premature ovarian failure show normal serum levels of FMRP; however, many show decreased levels of FMR1 mRNA.  Recently, a number of premutation carriers have been found to have a tremor-ataxia syndrome, termed Fragile X-associated tremor-ataxia syndrome (FXTAS) characterized by development of an intention tremor between 50 and 65 years of age, gait ataxia, global brain atrophy, and variable degrees of memory loss and dementia (Hagerman and Hagerman, 2002).  In this study, we compare cerebral and cerebellar activation during both motor and cognitive tasks in patients with the Fragile X premutation to that in normal age-matched controls.  The motor task is a simple stimulus-response protocol, requiring the subject to press four buttons, one with each finger of the dominant hand, when the appropriate stimulus appears.  The cognitive aspect of the study is divided into two components, math and working memory.  The mathematical component requires the subject to respond by pressing a button when a presented equation is true, while ignoring false equations.  In the working memory task, subjects are presented with an array of six letters, followed by a single letter, and asked to press a button if the single letter was in the array preceding it.  Activation is measured by fMRI-specific statistical parametric mapping and considered significant when the degree of activation in the test condition exceeds that of the control condition with a p-value less than 0.01.  As the syndrome’s characteristic clinical signs are commonly considered indicative of cerebellar disorders, we expect to see decreased cerebellar activation in the premutation subjects.  Additionally, the overall cognitive decline seen in FXTAS patients suggests that they will show an increased error rate on the cognitive tasks compared to control subjects.
 

IL-8 AND ITS ROLE IN ANDROGEN INDEPENDENCE OF PROSTATE CANCER CELL LINES

PSTP Student: Yu-Ming Chang
Mentor: Hsing-Jien Kung
Collaborators:  Evans, C

Prostate cancer (CaP) is the most prevalent cancer and the second most frequent cause of cancer related death among American men.  Although anti-androgen therapy results in cancer cell apoptosis during its growth both in vivo and in vitro, CaP eventually develops androgen independence and proliferates uninhibited despite treatment, thus leading to patient morbidity and mortality.  Interluekin-8 is a CXC chemokine initially identified as a regulator for the recruitment and trafficking of leukocytes to sites of inflammation.  Serum IL-8 levels have been shown to exhibit a two-fold increase in patients with metastatic CaP than those with localized disease and a 4-5-fold increase when compared to patients without prostatic disease or benign prostatic hyperplasia.

We are thus interested in IL-8 as a factor of androgen independence in CaP.  In our experiments, we used CWR22Rv1, an androgen sensitive but androgen independent cell line of xenographic origin as a model for CaP.  We tested our hypothesis by examining the growth rate of CWR22Rv1 via MTT proliferation assay in regular and androgen-free media with the addition of exogenous IL-8, casodex as an androgen receptor inhibitor, or both.  We also developed IL-8 overexpressing clones, verified by ELISA, and examined the growth rates of IL-8 clones with that of mock transfectants.  Lastly, we examined the effect of the addition of monoclonal anti-IL-8 antibody and/or casodex in both regular and androgen-free media.

In cell proliferation assays, we found that the addition of exogenous IL-8 resulted in a more rapid growth of CaP in androgen-free media.  Similarly, IL-8 transfectants of CWR22Rv1 grow more rapidly than mock transfectants in androgen-free media.  The addition of anti-IL-8 to IL-8 producing clones also resulted in slower growth.  We therefore conclude that IL-8 may support androgen independence of CaP.
 

POPULATION STRUCTURE AND MARKER CHARACTERISTICS IN EUROPEAN AMERICANS, MEXICAN AMERICANS, AMERINDIANS AND ASIANS

PSTP Student: Heather E. Collins-Schramm
Mentor:  Dr. Michael F. Seldin

Markers with large differences in allele frequencies between ethnicities provide ancestry information that can be applied to genetic studies.  These markers also provide an opportunity to examine population structure and relationships between populations. We identified over 100 biallelic Ancestry Informative Markers (AIMs) with large allele frequency differences between European Americans (EA) and Pima Amerindians from laboratory and database screens.  For 35 of these markers, Mayan, Yavapai and Quechuan Amerindians were genotyped and compared with EA and Pima allele frequencies.   Markers with large allele frequency differences between EA and one Amerindian tribe showed only small differences between the Amerindian tribes (e.g. the mean standard variance (f) for EA/Pima = 0.36, while Pima/Mayan = 0.02).  Examination of structure in individuals demonstrated a clear separation of subjects of European from those of Amerindian ancestry, and a remarkable similarity between individuals from disparate Amerindian populations.  The AIMs demonstrated the variation in ancestral composition of individual Mexican Americans, providing evidence of applicability in admixture mapping and in controlling for structure in association tests.  In addition, a high percentage of SNPs selected on the basis of large frequency differences between EA and Asian populations had large allele frequency differences between EA and Amerindians (e.g. 13/30 markers with EA/Japanese f>0.3 had EA/Pima f >0.30), suggesting an efficient method for greatly expanding AIMs for use in admixture mapping/structure analysis in Mexican Americans.  Together, these data provide additional support for the practical application of admixture mapping in the Mexican American population. 
 

A Quantitative Study on Microvascular Changes in Hypovolemia and Resuscitation Treatment

PSTP Student: Patricia L. Duong
Mentor: Dr. Anthony T. Cheung

In both human and veterinary medicine, resuscitation with allogenic blood has long been the mainstay in the treatment of acute and severe blood loss despite serious concerns associated with its use. To address these concerns, allogenic and xenographic, stroma-free, and ultrapurified artificial blood substitutes have been developed, with restoration of systemic function and oxygenation variables as the primary goal. However, there has been little emphasis on studying the effects of these products in the real-time microcirculation. In this study, microvascular changes in moderate hypovolemia and resuscitation treatment using blood substitutes [6% hetastarch and HB-200 (a hemoglobin-based oxygen carrier)] with autologous (shed) blood as control were studied. In addition, systemic function and oxygenation changes were monitored continuously for correlation. Nine animals were studied—after splenectomy, each animal was hemorrhaged (MAP reduced to 50 mmHg; ~40% blood loss=32-36 ml/kg) and randomly assigned to 3 resuscitation groups: hetastarch (n=3), HB-200 (n=3), and control (n=3). Computer-assisted intravital microscopy (CAIM) was used to videotape and objectively quantify microvascular changes in the conjunctival microcirculation during prehemorrhagic, posthemorrhagic, and resuscitation phases of the study. Prehemorrhagic microvascular characteristics were similar in all animals (diameter of venules=41±12?m, A:V ratio~1:2, red-cell velocity=0.5±0.3mm/s). All animals showed similar significant (P<0.01) posthemorrhagic changes: ~19% decrease in vessel diameter (diameter of venules=34±7?m), A:V ratio=extremely variable, and ~80% increase in red-cell velocity (0.9±0.5mm/s). Control blood resuscitation restored microvascular changes to prehemorrhagic baseline values (diameter=39±6?m, A:V ratio~1:2, red-cell velocity=0.6±0.4mm/sec). Hetastarch (non-oxygen carrier) and HB-200 (oxygen carrier) also restored microvascular changes to prehemorrhagic values in similar manner (Hetastarch diameter=38±7?m, A:V ratio~1:2, red-cell velocity=0.5±0.4mm/s; HB-200 diameter=38±3?m, A:V ratio~1:2, red-cell velocity=0.6±0.4mm/s) despite differences in oxygen-carrying capability. These results suggest that only volume replenishment alone (and not oxygen-carrying capability) is needed to treat moderate hypovolemia or that oxygenation measurements obtained by standard methods may not reflect tissue oxygenation levels.

THE ROLE OF PURINERGIC RECEPTORS IN THE EXERCISE PRESSOR REFLEX

PSTP Student:  Ramy Hanna
PSTP Mentor:  Marc Kaufman

The changes in autonomic function that accompany exercise maintain a homeostatic supply of oxygenated blood to exercising muscles and purge their vascular beds of the metabolic by-products of contraction.  The exercise pressor reflex is one mechanism thought to contribute to the elicitation of these autonomic adaptations. The sensory limb of this reflex arc is known to be comprised of group III and IV skeletal muscle afferents. The purpose of these studies was to better understand the nature of the metabolites activating group III and IV afferents. Specifically, we sought to determine what, if any, role adenosine 5’-triphosphate (ATP) played in evoking the exercise pressor reflex. Three consecutive questions were introduced to provide an experimental framework whereby we can determine this role. The first question asked was whether an exogenous ATP analog would elicit neurally-mediated increases in cardiovascular and ventilatory functions similar to those evoked by exercise. In the first branch of the studies, we provided experimental evidence that injection of a, ß-methylene ATP into the vascular supply of triceps surae muscles elicits a reflex pressor response that was mediated via P2X purinergic receptors. The second question asked whether endogenous ATP, released during static muscle contraction, would elicit increases in cardiovascular and ventilatory function like those seen with exercise. In the second branch of the studies, we provided data to show that P2 receptor blockade abolished the pressor responses to static contraction of the triceps surae muscles, to stretch of this muscle group and to a period of post-exercise circulatory occlusion. The third question asked if group III and IV muscle afferents would be responsive to exogenous ATP analogs as well as endogenous ATP released during muscle contraction. In the third branch of the studies, we presented results showing that the majority of group IV afferents from the triceps surae muscles responded to a, ß-methylene ATP, a P2X receptor agonist. This data supported our hypothesis that ATP elicits a reflex pressor response arising from contracting muscle by stimulating P2X receptors on group IV afferents. 

AMILORIDE TREATMENT PRODUCES DEATH IN MALIGNANT HUMAN GLIOMA CELL LINES

PSTP Student: Manu Hegde
Mentor: Fred Gorin
Collaborators: Roscoe J, Cala P

The type I sodium/hydrogen exchanger (NHE1) is a ubiquitously expressed transport protein involved in pH and volume regulation.  Previously, we reported that several human and rat malignant glioma cell lines maintain an elevated intracellular pH (pHi 7.2-7.5), compared with primary rat astrocytes (pHi 6.9-7.0), which can be attributed to persistent activation of NHE1 in glioma cells (McLean 2000).  In this study, high doses of the NHE1 inhibitor amiloride (= 500 µM) reduced glioma cell viability at 16 h, and produced extensive cell death by 48 h  (70-80%).  In contrast, primary astrocyte viability decreased by less than 20% after 24 h of high-dose amiloride treatment.  Modest and comparable increases in caspase-3 activities were observed in both astrocytes and glioma cells with amiloride treatment.  The inclusion of a pan-caspase inhibitory peptide (zVAD.fmk) partially reduced amiloride-associated glioma cell death at 72 h.  Cellular and nuclear volumes were increased in dead and dying glioma cells treated with amiloride, in contrast to the shrunken, classically apoptotic morphologies observed in staurosporine-treated cells.  These findings indicate amiloride produces largely necrotic cell death, with a possible caspase-dependent component at late times.  Neither lower doses of amiloride, the use of a more selective NHE1 inhibitor (cariporide), nor direct intracellular acidification of the glioma cells could replicate the selective glioma cell death produced by high dose amiloride.  Furthermore, alkaline buffering of glioma cell pHi in the presence of high dose amiloride failed to mitigate the drug’s cytotoxic effects.  These findings suggest that high-dose amiloride’s selective cytotoxicity to glioma cells is likely due to a mechanism independent of its inhibition of NHE1.

BIOMECHANICAL ANALYSIS OF THREE SURGICAL APPROACHES FOR LUMBAR CORPECTOMY USING ALLOGRAFT AND SYNTHETIC DEVICES

PSTP Student:  Phil Huang
Mentor:  Dr. Nesrin Sarigul-Klijn
Collaborators:  Hazelwood S, Gupta M

Fractures, tumors, bacterial infections and other diseases affecting vertebral bodies can result in either spinal instability and/or neural impingement.  For many of these conditions it is mandatory to reconstruct the destabilized spine after anterior decompression of the vertebral body, or vertebral corpectomy. Anterior bone grafting is often performed.  However, bone grafting alone does not provide spinal stability immediately after the surgery.  Therefore, either anterior or posterior spinal instrumentation, combined with anterior bone graft, has been advocated for the restoration of vertebral column stability.   To further improve patient care, it is critical to further evaluate a variety of instruments through biomechanical testing.  Since it is important to perform evaluations in the context of the natural healing abilities of soft tissue and bone we are developing an in vivo sheep model for analyzing interactions between fusions and anterior spinal instrumentation.  Lumbar spinal instability will be created in the animal model by a corpectomy procedure at a given level.  Next, the following current vertebral body replacement methods will be implemented in the animal model: 1) femoral ring allograft; 2) Titanium Mesh Cage; and 3) Expandable Vertebral Body Replacement Device. These strut implants will be used with a locking plate for anterior fixation.  Following an appropriate postoperative period, the animals will be sacrificed and their lumbar spines harvested.  This will allow for biomechanical, radiological, and histological assessment.  All anterior instrumentation will then be removed.  Testing only the vertebral bodies and the replacement for the corpectomized unit will serve as a true test of the fusion success of each graft/device.  Biomechanical testing parameters will include subjecting the segment to torsion, flexion/extension, and lateral bending.  Load versus deformation data will allow for the determination of torsional, axial, and flexural stiffnesses.  Another consideration will be the use of strain gauges attached to the surfaces of the vertebral bodies and implants to provide direct measurement of axial and flexural strains.  Stiffness and/or strain data can then be compared across the fused vertebral constructs to determine if one is superior to the others.  These studies should provide valuable insight for the design and development of the next generation of implants for spinal stabilization.

DIFFERENTIAL REGULATION OF NUCLEAR COREPRESSOR PROTEINS, SILENCING MEDIATOR OF RETIONIC ACID AND THYROID HORMONE RECEPTORS (SMRT) AND NUCLEAR HORMONE RECEPTOR COREPRESSOR (N-COR)

PSTP Student:  Brian Jonas
Mentor:  DR. Martin Privalsky

The two nuclear receptor corepressors, Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptors (SMRT) and its paralog Nuclear Hormone Receptor Corepressor (N-CoR), are evolutionarily conserved proteins involved in metazoan transcriptional regulation.  These proteins play critical roles in determining patterns of growth, differentiation, development, and homeostasis.  Regulation of corepressor proteins is of paramount importance to the cell since errors in transcriptional regulation are causal factors in several severe human diseases including Resistance to Thyroid Hormone, Acute Promyelocytic Leukemia and Mammary Cancer.  Classically, these corepresssor proteins are regulated by the binding of small, lipophilic hormones to nuclear hormone receptors such as the Thyroid Receptor (TR) and Retinoic Acid Receptor (RAR).  In addition, it is becoming clear that SMRT and N-CoR are regulated by other mechanisms including phosphorylation and ubiquitination leading to changes in receptor interaction, cellular relocalization and stability.  Furthermore, much of the current literature focuses on either SMRT or N-CoR often leading to generalizations regarding the parallel regulation of the other.  We, however, have evidence SMRT and N-CoR are regulated in a non-identical manner and it is likely this differential regulation will prove to be important in the overall physiology of corepressor function.  Using a variety of techniques, including mammalian two-hybrid anaylsis, transient transfection transcriptional assays, co-immunoprecipitation experiments and fluorescence microscopy, we have shown that Mitogen-Activated Protein Kinase (MAPK) pathway signaling regulates the ability of N-CoR to interact with TR in a differential manner compared to SMRT.  Furthermore, previously published work showing a translocation of SMRT from the nucleus to the cytoplasm in the presence of MAPK signaling does not occur in the case of N-CoR.  Finally, we have also observed that the effects of kinase signaling on the steady-state stability of SMRT and N-CoR differs in many of our assays.
 

REGULATION OF G PROTEIN DEACTIVATION IN ROD PHOTORECEPTORS

PSTP Student: Claudia Krispel
Mentor: Dr. Marie Burns

Temporal regulation of G protein cascades is important for proper functioning of neurons.  In rod photoreceptors, the deactivation of the cascade determines the time course and sensitivity of the response, and modulation of these deactivation steps mediates light adaptation.   One important step in turning off the response to light is deactivation of the G protein/Effector complex.  One factor necessary for G protein / Effector deactivation is RGS9, a catalyst of GTP hydrolysis.  RGS9 exists in a complex with an orphan G protein beta subunit, G?5, the function of which has been unclear up until recently.  Using suction electrodes to record photoreceptor responses to brief flashes of light, we have shown that responses from G?5 knockout rods are significantly slowed, and are identical to responses from RGS9 knockout rods.  From this, we concluded that G?5 is necessary for proper hydrolysis of GTP.  In addition, we found that rods lacking G?5 do not adapt normally to steady light.  This led us to wonder if perhaps other mechanisms regulating the G protein/effector deactivation are important in adaptation of rod photoreceptors.  We have recorded responses of individual rod photoreceptors to brief flashes of light before and immediately after several minutes of a bright background light and have found that immediately after removal of this adapting light, the duration of the rod’s flash response is significantly shorter, and returns to its original form over a period of minutes.  Experiments on RGS9 knockout rods suggest that speeding of the G protein / Effector deactivation underlies this form of adaptation.  Elucidating this mechanism is essential for understanding phototransduction, and likely will be important for understanding many other signaling cascades.

ULTRASTRUCTURAL ANALYSIS OF TRANSIENT MOSSY FIBER PROJECTIONS IN THE HIPPOCAMPUS OF CD-1 MICE

PSTP Student: Lawrence Low
Mentor:  Dr. Hwai-Jong Cheng

The ultrastructural characteristics of transient projections of the central nervous system have not been well characterized.  Using immuno-based transmission electron microscopy to specifically label with anti-calbindin antibody both the main bundle (MB) permanent and the infrapyramidal bundle (IPB) transient mossy fibers in the developing hippocampus of CD-1 mice aged postnatal day 15-35, we sought to examine the transient projections as they were forming—for synapse formation—and as they were being pruned back—for synapse elimination and signs of degeneration.  The MB fibers that form permanent synapses were used as a control for comparison.  MB fibers establish permanent synapses that become increasingly more complex with age as expected.  On the other hand, the morphological changes occurring at axon terminals during the development of transient IPB projections is dramatically reversed.  We discovered that by age P15 when the IPB is longest, a majority of the labeled mossy fibers form synapses resembling morphological characteristics that were mostly intermediate in complexity when compared to the morphologies of MB fibers.  At P25 and P35 when the IPB is pruning back and pruned back to its mature form, respectively, the complexity of the fiber terminals is dramatically decreased and the number of synaptic contacts are also decreased significantly as well.  Furthermore, no signs of degeneration in the IPB were observed at all stages of development.  The results support the views that synapse formation occurs during development of transient aberrant projections and degeneration is not a major pathway for axonal pruning in the central nervous system of vertebrates.

TNF-a DECREASES AKT PROTEIN LEVELS IN 3T3-L1 ADIPOCYTES:  INVOLVEMENT OF THE CASPASE AND UBIQUITIN/PROTEOSOME PATHWAYS

PSTP Student:  Edward A. Medina
Mentor:  Kent L. Erickson
Collaborator:  Goldkorn T

Tumor necrosis factor-alpha (TNF-a? impairs insulin signaling in adipocytes.  Akt (protein kinase B) is a crucial signaling mediator for insulin.  In the present study, we examined the posttranslational mechanisms by which short-term (< 6 h) exposure to TNF-a? decreases Akt expression in 3T3-L1 adipocytes.  TNF-a? increased the ubiquitination of Akt1 and Akt2, and decreased the protein levels of Akt1 and Akt2.  Two 40-50 kD Akt1 fragments were observed, but not for Akt2.  The caspase inhibitor Boc-D-FMK markedly suppressed these effects of TNF-a?.  The proteosome inhibitor MG132 modestly attenuated the decline in Akt1 and completely protected Akt2.  Adipocytes preexposed to either TNF-a?  for 5 h and then stimulated with insulin for 30 min, or exposed to both insulin and TNF-a? for 4 h, exhibited decreased levels of Akt, phosphorylated Akt, and Mdm2 phosphorylated at Ser166, which is a specific site for phosphorylation by Akt.  Caspase and proteosome inhibition attenuated these effects of TNF-a?.  These results suggest that TNF-a?  induces the caspase-dependent degradation of Akt via two mechanisms: the cleavage of Akt1, and the ubiquitination of Akt1 and Akt2, which results in their degradation via the 26S proteosome.  Furthermore, caspase- and proteosome-mediated degradation of Akt due to TNF-a? exposure impairs Akt-dependent insulin signaling

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A SUMMARY OF FIVE STEREOTACTIC NEUROSURGERY PATIENTS IN MADRAS, INDIA

PSTP Student:  Noah Merin
Mentor:  Dr. David Amaral

Over the past two years, we have been engaged in identifying patients in India who have undergone bilateral amygdalectomy.  We have now found and performed MRI on five patients with purported bilateral lesions.  Of these patients, only one has bilateral lesions restricted to the amygdala.  The remaining patients have either unilateral amygdala lesions or significant damage to other brain structures, including the insular cortex, ventrolateral prefrontal cortex, white matter dorsolateral to amygdala, and entorhinal cortex deep to amygdala.  These findings suggest that the lesion techniques and surgical localization procedures were imprecise, and imply that we may need to scan significantly more patients than we anticipated in order to generate a cohort of patients with bilateral amygdala lesions.

THE HUMAN HOMOLOG OF ARABIDOPSIS DE-ETIOLATED 1 IS A SUBSTRATE RECEPTOR FOR A MULTISUBUNIT C-JUN UBIQUITIN LIGASE

PSTP Student:  Ingrid Wertz
Mentor:  Dr. Vishva M. Dixit
Collaborators:  O’Rourke KM, Zhang Z, Dornan D, Arnott D, Deshaies RJ

Arabidopsis DET1 and COP1 are highly conserved proteins, with homologs found in vertebrates, invertebrates, and Dictyostelium.  In plants, DET1 and COP1 negatively regulate light-activated transcription factors.  However their precise mechanisms of action, and their purpose in other species, is unknown.   We find that human DET1 (hDET) and human COP1 (hCOP) associate with each other and assemble into a multi-subunit ubiquitin ligase complex containing DDB1, CUL4A, and ROC1.  In so doing, hDET and hCOP cooperate to ubiquitinate and degrade the transcription factor c-jun.  The reconstituted complex ubiquitinated c-jun in vitro, and reducing expression of any subunit with RNAi stabilized c-jun and increased c-jun-activated transcription.  Our findings identify a novel c-jun ubiquitin ligase and define specific functions for hDET and hCOP, which may explain how DET1 and COP1 regulate photomorphogenesis.

 

LONG-TERM CORRECTION OF HYPERGLYCEMIA IN DIABETIC MICE AFTER IMPLANTATION OF CULTURED HUMAN CELLS DERIVED FROM FETAL PANCREAS

PSTP Student:  Fred Wu
Mentor:  Dr. Jerry Powell

Type I diabetes is characterized by destruction of insulin producing beta-islet cells in the pancreas resulting in hyperglycemia and associated morbidity. The successful treatment of diabetes by transplanted islets has resulted in increased efforts to identify methods to increase islet availability. One approach is to identify and expand islet precursor cells able to later differentiate into functional endocrine cells. Human fetal pancreas (HFP) was used as an enriched source of beta-islet precursor cells. A population of cytokeratin19-, vimentin+, insulin-, glucagon-, nestin+ cells was established and cultured for over 20 population doublings. During this time, the cells maintained the ability to form cell aggregates when stimulated by growth on poly-D-lysine (PDL) coated surfaces. These cells were then evaluated for differentiation potential using in vivo function as a surrogate marker for the presence of differentiated precursor cells. Severe combined immunodeficient (SCID) mice were implanted with PDL induced cell aggregates (PCA) or monolayer cells. Over the next four weeks, mice implanted with PCA produced detectable amounts of human c-peptide after glucose tolerance testing, whereas control mice and mice implanted with monolayer cells did not.

In addition, PCA implanted animals maintained normoglycemia, suggesting that insulin production was being properly regulated. Streptozotocin induced diabetic SCID mice (glucose concentrations > 300) implanted with PDL induced cell aggregates (PCA) were able to maintain glucose concentrations below 200 mg/dl for over 70 days (n = 5). In addition, human c-peptide levels responsive to glucose tolerance testing was detected in implanted animals, but not in control animals. These findings show that a population of HFP derived cells 1)
can be cultured and expanded in vitro, 2) can maintain the ability to differentiate into beta-islet-like cells that express detectable amounts of human c-peptide, and 3) can correct hyperglycemia in a mouse model of
diabetes. Further improvements in isolation, identification, culture, and differentiation of human pancreas derived beta-cell precursors may one day help to provide a novel source of islets for use in transplantation therapy to
cure type I diabetes.