Candace L. Kerr

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Expression of pluripotent stem cell markers in the human fetal testis.

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). However, the developmental potency of these cells in the fetal gonad still remains elusive. Thus, this study provides a comprehensive analysis of pluripotent and germ cell marker expression in human fetal testis 7–15 weeks postfertilization (pF) and compares this expression to their ability to derive EGCs. Although the majority of germ cells expressed stem cell markers stage‐specific embryonic antigen (SSEA) 1, SSEA4, EMA‐1, and alkaline phosphatase, only a small percentage of those (<1%) expressed OCT4, CKIT, and NANOG. Specifically, the number of OCT4+/CKIT+/NANOG+ cells significantly increased in the developing cords during weeks 7–9, followed by a gradual decline into week 15 pF. By week 15 pF, the remaining OCT4+/CKIT+/NANOG+ cells were found in the cords surrounding the periphery of the testis, and the predominant germ cells, CKIT+ cells, no longer expressed OCT4 or NANOG. Based on morphology and early germ cell marker expression, including VASA, PUM2, and DAZL, we suggest these cells are mitotically active gonocytes or prespermatogonia. Importantly, the number of OCT4+ cells correlated with an increase in the number of EGC colonies derived in culture. Interestingly, two pluripotent markers, Tra‐1–60 and Tra‐1–81, although highly expressed in EGCs, were not expressed by PGCs in the gonad. Together, these results suggest that PGCs maintain expression of pluripotent stem cell markers during and after sexual differentiation of the gonad, albeit in very low numbers.

Expression of pluripotent stem cell markers in the human fetal ovary

BACKGROUND Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). METHODS In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro. RESULTS Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad. CONCLUSIONS These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.

MicroRNA Expression Profiling of Oligodendrocyte Differentiation from Human Embryonic Stem Cells

Background Cells of the oligodendrocyte (OL) lineage play a vital role in the production and maintenance of myelin, a multilamellar membrane which allows for saltatory conduction along axons. These cells may provide immense therapeutic potential for lost sensory and motor function in demyelinating conditions, such as spinal cord injury, multiple sclerosis, and transverse myelitis. However, the molecular mechanisms controlling OL differentiation are largely unknown. MicroRNAs (miRNAs) are considered the “micromanagers” of gene expression with suggestive roles in cellular differentiation and maintenance. Although unique patterns of miRNA expression in various cell lineages have been characterized, this is the first report documenting their expression during oligodendrocyte maturation from human embryonic stem (hES) cells. Here, we performed a global miRNA analysis to reveal and identify characteristic patterns in the multiple stages leading to OL maturation from hES cells including those targeting factors involved in myelin production. Methodology/Principal Findings We isolated cells from 8 stages of OL differentiation. Total RNA was subjected to miRNA profiling and validations preformed using real-time qRT-PCR. A comparison of miRNAs from our cultured OLs and OL progenitors showed significant similarities with published results from equivalent cells found in the rat and mouse central nervous system. Principal component analysis revealed four main clusters of miRNA expression corresponding to early, mid, and late progenitors, and mature OLs. These results were supported by correlation analyses between adjacent stages. Interestingly, the highest differentially-expressed miRNAs demonstrated a similar pattern of expression throughout all stages of differentiation, suggesting that they potentially regulate a common target or set of targets in this process. The predicted targets of these miRNAs include those with known or suspected roles in oligodendrocyte development and myelination including C11Orf9, CLDN11, MYTL1, MBOP, MPZL2, and DDR1. Conclusions/Significance We demonstrate miRNA profiles during distinct stages in oligodendroglial differentiation that may provide key markers of OL maturation. Our results reveal pronounced trends in miRNA expression and their potential mRNA target interactions that could provide valuable insight into the molecular mechanisms of differentiation.

Efficient differentiation of human embryonic stem cells into oligodendrocyte progenitors for application in a rat contusion model of spinal cord injury

ABSTRACT This study utilized a contusion model of spinal cord injury (SCI) in rats using the standardized NYU-MASCIS impactor, after which oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (ESC) were transplanted into the spinal cord to study their survival and migration route toward the areas of injury. One critical aspect of successful cell-based SCI therapy is the time of injection following injury. OPCs were injected at two clinically relevant times when most damage occurs to the surrounding tissue, 3 and 24 hours following injury. Migration and survivability after eight days was measured postmortem. In-vitro immunofluorescence revealed that most ESC-derived OPCs expressed oligodendrocyte markers, including CNPase, GalC, Olig1, O4, and O1. Results showed that OPCs survived when injected at the center of injury and migrated away from the injection sites after one week. Histological sections revealed integration of ESC-derived OPCs into the spinal cord with contusion injury without disruption to the parenchyma. Cells survived for a minimum of eight days after injury, without tumor or cyst formation. The extent of injury and effect of early cell transplant was measured using behavioral and electrophysiological assessments which demonstrated increased neurological responses in rats transplanted with OPCs compared to controls.

Mice That Express Enzymatically Inactive Cathepsin L Exhibit Abnormal Spermatogenesis

Abstract The finding of large, stage-specific changes in secretion of procathepsin L by rat Sertoli cells has led to the hypothesis that this proenzyme promotes the survival, replication, or differentiation of spermatogenic cells. Experiments described herein used a mouse model to test this hypothesis. To prove that mice are appropriate for this purpose, we first demonstrate that mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as rat Sertoli cells and they also secrete procathepsin L. To test whether catalytically active cathepsin L is required for normal spermatogenesis, we examined the testes of 110- to 120-day-old furless mice, which express catalytically inactive cathepsin L. Morphologic examination of testes of furless mice revealed both normal and atrophic seminiferous tubules. Enumeration of atrophic tubules in furless and control mice demonstrates that lack of functional cathepsin L results in a 12-fold increase in seminiferous tubule atrophy. To determine whether lack of functional cathepsin L affects the production of male germ cells in apparently normal, nonatrophic tubules, we compared numbers in control and furless mice of preleptotene spermatocytes, pachytene spermatocytes, and round spermatids per Sertoli cell. Results demonstrate that the lack of functional cathepsin L causes a 16% reduction in formation of preleptotene spermatocytes and a 25% reduction in differentiation of these cells into pachytene spermatocyte. These results suggest that procathepsin L either directly or indirectly has two distinct functions in the testis. This proenzyme prevents atrophy of seminiferous tubules and promotes the formation of preleptotene spermatocytes and the differentiation of these meiotic cells into pachytene spermatocytes.

HMGA1 Reprograms Somatic Cells into Pluripotent Stem Cells by Inducing Stem Cell Transcriptional Networks

Background Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. Methods/Principal Findings We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC – OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. Conclusions Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Characterization of graded multicenter animal spinal cord injury study contusion spinal cord injury using somatosensory-evoked potentials.

Study Design. Electrophysiological analysis using somatosensory-evoked potentials (SEPs) and behavioral assessment using Basso, Beattie, Bresnahan (BBB) scale were compared over time for graded Multicenter Animal Spinal Cord Injury Study (MASCIS) contusion spinal cord injury (SCI). Objective. To study the SEP responses across different contusion injury severities and to compare them with BBB scores. Summary of Background Data. For any SCI therapy evaluation, it is important to accurately and objectively standardize the injury model. The graded MASCIS contusion injuries on dorsal spine have been standardized using BBB, which is subjective and prone to human errors. Furthermore, dorsal pathway disruption does not always produce locomotor deficits. SEP monitoring provides an advantage of providing a reliable and objective assessment of the functional integrity of dorsal sensory pathways. Methods. Four groups of Fischer rats received contusion at T8 using New York University (NYU)-MASCIS impactor from impact heights of 6.25 mm (mild), 12.5 mm (moderate), 25 mm (severe), or 50 mm (very severe). The control group underwent laminectomy only. SEP and BBB recordings were performed once before injury, and then weekly for up to 7 weeks. Results. Graded levels of injury produced concomitant attenuations in hindlimb SEP amplitudes. Following injury, 25 and 50 mm groups together differed significantly from 12.5 and 6.25 mm groups (P < 0.01). From week 5, differences between 12.5 and 6.25 mm groups also became apparent (P < 0.01), which showed significant electrophysiological improvement. However, no significant differences were observed between 25 and 50 mm groups, which showed negligible electrophysiological recovery. Although comparable differences between different groups were also detected by BBB after injury (P < 0.001), BBB was less sensitive in detecting any improvement in 6.25 and 12.5 mm groups. Conclusion. SEP amplitudes and BBB scores decrease corresponding to increase in injury severity, however, these show different temporal patterns of recovery. These results demonstrate the utility of SEPs in conjunction with BBB, to monitor therapeutic interventions in SCI research.

Temporal analysis of neural differentiation using quantitative proteomics

The ability to derive neural progenitors, differentiated neurons and glial cells from human embryonic stem cells (hESCs) with high efficiency holds promise for a number of clinical applications. However, investigating the temporal events is crucial for defining the underlying mechanisms that drive this process of differentiation along different lineages. We carried out quantitative proteomic profiling using a multiplexed approach capable of analyzing eight different samples simultaneously to monitor the temporal dynamics of protein abundance as human embryonic stem cells differentiate into motor neurons or astrocytes. With this approach, a catalog of approximately 1200 proteins along with their relative quantitative expression patterns was generated. The differential expression of the large majority of these proteins has not previously been reported or studied in the context of neural differentiation. As expected, two of the widely used markers of pluripotency, alkaline phosphatase (ALPL) and LIN28, were found to be downregulated during differentiation, while S-100 and tenascin C were upregulated in astrocytes. Neurofilament 3 protein, doublecortin and CAM kinase-like 1 and nestin proteins were upregulated during motor neuron differentiation. We identified a number of proteins whose expression was largely confined to specific cell types, embryonic stem cells, embryoid bodies and differentiating motor neurons. For example, glycogen phosphorylase (PYGL) and fatty acid binding protein 5 (FABP5) were enriched in ESCs, while beta spectrin (SPTBN5) was highly expressed in embryoid bodies. Karyopherin, heat shock 27 kDa protein 1 and cellular retinoic acid binding protein 2 (CRABP2) were upregulated in differentiating motor neurons but were downregulated in mature motor neurons. We validated some of the novel markers of the differentiation process using immunoblotting and immunocytochemical labeling. To our knowledge, this is the first large-scale temporal proteomic profiling of human stem cell differentiation into neural cell types highlighting proteins with limited or undefined roles in neural fate.

Potential long-term benefits of acute hypothermia after spinal cord injury: Assessments with somatosensory-evoked potentials*

Objective: Neuroprotection by hypothermia has been an important research topic over last two decades. In animal models of spinal cord injury, the primary focus has been assessing the effects of hypothermia on behavioral and histologic outcomes. Although a few studies have investigated electrophysiological changes in descending motor pathways with motor-evoked potentials recorded during cooling, we report here hypothermia induced increased electrical conduction in the ascending spinal cord pathways with somatosensory-evoked potentials in injured rats. In our experiments, these effects lasted long after the acute hypothermia and were accompanied by potential long-term improvements in motor movement. Design: Laboratory investigation. Setting: University medical school. Subjects: Twenty-one female Lewis rats. Interventions: Hypothermia. Measurements and Main Results: All animals underwent spinal cord contusion with the NYU-Impactor by a 12.5-mm weight drop at thoracic vertebra T8. A group (n = 10) was randomly assigned for a systemic 2-hr hypothermia episode (32 ± 0.5°C) initiated approximately 2.0 hrs postinjury. Eleven rats were controls with postinjury temperature maintained at 37 ± 0.5°C for 2 hrs. The two groups underwent preinjury, weekly postinjury (up to 4 wks) somatosensory-evoked potential recordings and standard motor behavioral tests (BBB). Three randomly selected rats from each group were euthanized for histologic analysis at postinjury day 3 and day 28. Compared with controls, the hypothermia group showed significantly higher postinjury somatosensory-evoked potential amplitudes with longer latencies. The BBB scores were also higher immediately after injury and 4 wks later in the hypothermia group. Importantly, specific changes in the Basso, Beattie, Bresnahan scores in the hypothermia group (not seen in controls) indicated regained functions critical for motor control. Histologic evaluations showed more tissue preservation in the hypothermia group. Conclusions: After spinal cord injury, early systemic hypothermia provided significant neuroprotection weeks after injury through improved sensory electrophysiological signals in rats. This was accompanied by higher motor behavioral scores and more spared tissue in acute and postacute periods after injury.