Scientific Resources

Peripheral blood was collected from a clinically diagnosed 79-year old male sporadic Parkinson's disease patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model can be used to study the mechanism of sporadic Parkinson's disease and to test new drugs. Resource Table. View Publication

Peripheral blood was collected from a clinically diagnosed 72-year old male patient with later onset Alzheimer's disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for studying the pathological mechanism of Alzheimer's disease. View Publication

Peripheral blood was collected from a clinically diagnosed 60-year old female patient with multiple schwannoma. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma. View Publication

The heart wall tissue,or the myocardium,is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally,current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells,lack of a 3D biomimetic environment,and the use of non-human cells. In this study,we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels,to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining,genetic,and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality,and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment,and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days,and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line,essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner. View Publication

Urine resource cells were collected from a 59-year-old female patient with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids carrying Oct4,Sox2,Klf4 and miR-302-367. The patient sustained a heterozygous GtextgreaterT transition mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on the obtained iPSC lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression,as well as their abilities for differentiating into three germ layers. This cell line provides an ideal model for studying MEN1. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with N551K variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model can complement in vivo PD models for pathophysiological studies and drug screening. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 72-year old female Parkinson's disease (PD) patient with R1398H variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model provides a good platform for studying the mechanism of PD,and also for drug testing and gene therapy studies. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 59-year old male Parkinson's disease (PD) patient with R1628P variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model will provide a good resource for further pathophysiological studies of PD. View Publication

Human fibroblast cells collected from a 3-year old,female Rett Syndrome patient with a 32bp deletion in the X-linked MECP2 gene were obtained from the Coriell Institute. Fibroblasts were reprogrammed to iPSC cells using a Sendai-virus delivery system expressing human KOSM transcription factors. Cell-line pluripotency was demonstrated by gene expression,immunocytochemistry,in-vitro differentiation trilineage capacity and was of normal karyotype. Interestingly,subsequent clones retained the epigenetic memory of the parent fibroblasts allowing for the segregation of wild-type and mutant expressing clones. This MECP2 mutant expressing clone may serve as a model for investigating MECP2 reactivation in Rett's Syndrome. View Publication

In the field of hematopoietic regeneration,deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells,remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines,UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here,using a serum-free,feeder-free,and chemically defined induction protocol,we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34(+)CD45(+) derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol,the derived hematopoietic progenitors failed to engraft in NOG mice,indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols,UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro. View Publication

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect,we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further,we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected,an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also,cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast,allowing cells to rest overnight in whole blood prior to stimulation through CD28,lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25),followed a similar pattern,with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. View Publication

OBJECTIVE Vitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However,the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS,we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis. METHODS Human T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls. RESULTS An alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%,interquartile range 19.2%) compared to controls (18.75%,9.5%),p-valuetextless0.05. Moreover,a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-valuetextless0.001). CONCLUSIONS An alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis,and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis. View Publication