Kirby L. Johnson

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

Microchimerism of presumed fetal origin in thyroid specimens from women: a case-control study

BACKGROUND Some so-called autoimmune diseases in women might be alloimmune and represent a chronic graft-versus-host response attributable to transplacentally acquired fetal cells. Thyroid disease is more common in women than men, and post partum exacerbation of thyroiditis is common. Our aim was to investigate whether there is an association between fetal cell microchimerism and thyroid disease in women. METHODS Surgical specimens were obtained from 29 women who underwent thyroidectomy for various thyroid disorders. Control specimens were taken from clinically and histologically normal thyroids obtained at necropsy from eight women who died from unrelated conditions. Medical records and pregnancy histories were reviewed. Fluorescence in-situ hybridisation analysis was done with probes specific for X and Y chromosomes. Slides were examined with a fluorescence microscope to detect the presence of male cells-with one X and one Y signal in the nucleus-among maternal cells containing two X signals. FINDINGS Male cells were seen in thyroid sections from 16 patients but not in those from controls (p=0.01). Male cells (1-165 per slide) were seen individually or in clusters in all thyroid diseases and were not restricted to inflammatory thyroid diseases. In one patient with a progressively enlarging goitre, we noted fully differentiated male thyroid follicles closely attached to and indistinguishable from the rest of the thyroid. INTERPRETATION Our findings suggest a relation between fetal cell microchimerism and thyroid disease. Furthermore, fetal stem cells might be capable of differentiation into mature thyroid follicles in their mothers with favourable environmental and developmental factors.

Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis.

OBJECTIVE The realization that fetal cells pass into the maternal circulation and can survive for many years has raised the question of whether fetal microchimerism can cause subsequent disease in the mother. Available data suggest that fetal-maternal transfusion may be related to some autoimmune diseases, notably systemic sclerosis (SSc). The goal of the current work was to identify and quantify tissue-specific fetal microchimerism in women with SSc. METHODS We analyzed multiple tissue specimens obtained at autopsy from women with SSc as well as women who had died of causes unrelated to autoimmunity, using fluorescence in situ hybridization to detect the presence of male cells in women with sons. Tissues analyzed included adrenal gland, heart, intestine, kidney, liver, lung, lymph node, pancreas, parathyroid, skin, and spleen. RESULTS Male cells were observed in tissue from at least 1 site in each woman with SSc and were found most frequently in spleen sections. After spleen, male cells were observed most frequently in lymph node, lung, adrenal gland, and skin tissue. The only tissue type in which male cells were not seen in any patient was pancreatic tissue. Male cells were not observed in tissue from women who had died of causes unrelated to autoimmunity. CONCLUSION The results of this study suggest that fetal cells migrate from the peripheral circulation into multiple organs in women with SSc. All of the women studied had previously given birth to sons, so it is likely that these cells are of fetal origin. While the relevance of this finding to the pathogenesis of SSc remains to be elucidated, the presence of fetal cells in internal organs suggests that they could play a role in disease pathogenesis and that they may preferentially sequester in the spleen. The presence of these male cells may also be a result of disease, possibly through the migration of terminally differentiated and/or progenitor cells to areas of tissue damage.

Microchimerism in a female patient with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a serious multisystem disease that has a striking propensity to affect women. The cause of SLE remains elusive. Fetomaternal cell trafficking, or the passage of fetal cells into the maternal circulation, is now a well-established phenomenon. In addition, fetal cells have been implicated in the development of preeclampsia and in the pathogenesis of scleroderma. We undertook this study to determine whether fetomaternal cell trafficking might also be involved in pathogenic processes in SLE. Fluorescence in situ hybridization analysis was performed using X and Y chromosome-specific probes on affected and unaffected tissue obtained at autopsy from a woman who had previously given birth to 2 males and who had died of complications of SLE. The goal of the analysis was to detect the presence of male cells of putative fetal origin. Male cells were found in every histologically abnormal tissue type that was examined, but were not found in histologically normal tissue. These data suggest that fetal cells may be associated with SLE. It is unclear whether their presence may be related to disease causation, an effect of disease progression, or unrelated to disease pathology. However, this case study is an important step toward understanding the potential relationship between fetomaternal cell trafficking and SLE pathology.

Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.

Cervical cancer and microchimerism

OBJECTIVE To determine whether microchimerism is involved in the pathogenesis or progression of cervical cancer. METHODS Cervical tissue was obtained from eight women who had at least one live-born son and who underwent radical hysterectomy after a diagnosis of cervical cancer. Control tissue was obtained from four women without cervical cancer who had at least one live-born son and from three women with cervical cancer and no male births. Tissue sections were analyzed with fluorescence in situ hybridization for the presence of fetal cells, defined by an X and Y chromosome. Immunolabeling was used to determine the phenotype of the presumed fetal cells. RESULTS Male cells were found in cervical tissue from all four patients for whom large sections (approximately 1.5 x 2 cm) were analyzed. Only one male cell was found in two of the four patients for whom small biopsy specimens (approximately 0.1 x 0.5 cm) were analyzed. No male cells were found in tissue specimens from controls, whether they were small or large sections. In immunolabeling studies, eight of 18 male cells from one patient were CD45-positive and nine of 37 male cells from two patients were cytokeratin-positive. No cells were positive for both markers. CONCLUSION Cervical cancer might be associated with microchimerism, possibly from fetomaternal cell trafficking. These results further expand the potential relationship between microchimerism and disease in women.

Microarray Analysis of Cell-Free Fetal DNA in Amniotic Fluid: a Prenatal Molecular Karyotype

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

A review of fluorescence in situ hybridization (FISH): current status and future prospects.

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.

Gene expression analysis in pregnant women and their infants identifies unique fetal biomarkers that circulate in maternal blood

The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Students t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications.

Increased Death of Adipose Cells, a Path to Release Cell‐Free DNA Into Systemic Circulation of Obese Women

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell‐free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.