Generalized joint hypermobility and risk of pelvic girdle pain in pregnancy: does body mass index matter?

BACKGROUND: Women with generalized joint hypermobility may be at increased risk of pregnancy-related pelvic girdle pain, but evidence is inconclusive. OBJECTIVES: In this prospective cohort study of 283 pregnant women in Norway, we aimed to study the association of generalized joint mobility with pelvic girdle pain, and to evaluate if pre-pregnancy body mass index was a modifier of the association. METHODS: Generalized joint hypermobility was defined as a score of ≥5/9 positive tests on the Beighton score measured in early pregnancy. Primary outcome was evening pain intensity in gestation week 30, measured by a 100 mm visual analogue scale. We applied linear regression analyses to estimate age-adjusted unstandardized beta coefficients. RESULTS: Evening pain intensity was similar among women with Beighton score ≥ 5/9 and women with Beighton score < 5/9 (age-adjusted mean difference 2.8 mm; 95% CI: -9.2 to 14.9 mm). Women with Beighton score ≥ 5/9 and pre-pregnancy body mass index ≥ 25 kg/m2, reported higher evening pain than women with Beighton score < 5/9 and pre-pregnancy body mass index <25 kg/m2 (age-adjusted mean difference 28.7 mm; 95% CI: 14.3-43.1 mm). CONCLUSIONS: Overall, evening pain intensity was similar among pregnant women with and without generalized joint hypermobility. However, women with a combination of generalized joint hypermobility and body mass index ≥25 kg/m2 reported higher evening pain compared to women with normal joint mobility and body mass index <25 kg/m2, suggesting that body mass index may modify the association. The estimates could be imprecise due to the small study sample, and our findings should be interpreted with caution.

Sexuality and mood changes in women with persistent pelvic girdle pain after childbirth: a case-control study.

BACKGROUND: Pelvic girdle pain is a common problem during pregnancy. For most women, the symptoms cease within the first 3-6 months of giving birth, but in some women the pain persists. In this study we investigate the sexuality and frequency of depressive symptoms in women with persistent pelvic girdle pain after childbirth and in healthy women. METHODS: We conducted a case-control study of women with persistent pelvic girdle pain after childbirth and a control group of healthy women. The frequency of depressive symptoms and sexuality were measured using the self-rating version of the Montgomery-Asberg Depression Rating Scale and the McCoy Female Sexuality Questionnaire. RESULTS: Forty-six women with persistent pelvic girdle pain and thirty-nine healthy women were enrolled. The frequency of depressive symptoms and the total score on female sexuality did not differ between the groups. However, pain during intercourse was more frequent (P < 0.001) in women with persistent pelvic girdle pain and caused them to avoid sexual intercourse frequently (P < 0.001). In multiple linear regression a higher frequency of depressive symptoms was reversely correlated with a lower score on female sexuality (ß = - 0,41, p < 0,001 95% CI -0,6 - -0,22) This association remained after adjusting for obstetric variables and individual characteristics. CONCLUSION: Depressive symptoms and female sexuality were similar between women with persistent pelvic girdle pain after childbirth and healthy controls. However, pain during intercourse and avoidance of sexual intercourse were more frequent among women with pelvic girdle pain.

Early dorsomedial tissue interactions regulate gyrification of distal neocortex.

The extent of neocortical gyrification is an important determinant of a species' cognitive abilities, yet the mechanisms regulating cortical gyrification are poorly understood. We uncover long-range regulation of this process originating at the telencephalic dorsal midline, where levels of secreted Bmps are maintained by factors in both the neuroepithelium and the overlying mesenchyme. In the mouse, the combined loss of transcription factors Lmx1a and Lmx1b, selectively expressed in the midline neuroepithelium and the mesenchyme respectively, causes dorsal midline Bmp signaling to drop at early neural tube stages. This alters the spatial and temporal Wnt signaling profile of the dorsal midline cortical hem, which in turn causes gyrification of the distal neocortex. Our study uncovers early mesenchymal-neuroepithelial interactions that have long-range effects on neocortical gyrification and shows that lissencephaly in mice is actively maintained via redundant genetic regulation of dorsal midline development and signaling.

Roof Plate-Derived Radial Glial-like Cells Support Developmental Growth of Rapidly Adapting Mechanoreceptor Ascending Axons.

Spinal cord longitudinal axons comprise some of the longest axons in our body. However, mechanisms that drive this extra long-distance axonal growth are largely unclear. We found that ascending axons of rapidly adapting (RA) mechanoreceptors closely abut a previously undescribed population of roof plate-derived radial glial-like cells (RGLCs) in the spinal cord dorsal column, which form a network of processes enriched with growth-promoting factors. In dreher mutant mice that lack RGLCs, the lengths of ascending RA mechanoreceptor axon branches are specifically reduced, whereas their descending and collateral branches, and other dorsal column and sensory pathways, are largely unaffected. Because the number and intrinsic growth ability of RA mechanoreceptors are normal in dreher mice, our data suggest that RGLCs provide critical non-cell autonomous growth support for the ascending axons of RA mechanoreceptors. Together, our work identifies a developmental mechanism specifically required for long-range spinal cord longitudinal axons.

Anatomical landmarks of the intra-pelvic side-wall as sources of pain in women with and without pregnancy-related chronic pelvic pain after childbirth: a descriptive study.

BACKGROUND: Chronic pelvic pain (CPP) affects 15-24% of women and can have a devastating impact on quality of life. Laparoscopy is often used in the investigation, although in one third of the examinations there is no visible pathology and the women may be dismissed without further investigation. Also, the contribution of skeletal, muscular, periosteal and ligamentous tissues to CPP remains to be further elucidated. The objective of the present study was to compare pain intensity provoked from anatomical landmarks of the intra-pelvic side-wall in women with pregnancy-related CPP after childbirth and women without such pain. METHODS: This is a descriptive study of 36 non-randomly selected parous women with CPP after childbirth and 29 likewise selected parous women after childbirth without CPP. Pain was determined by questionnaire and clinical examination. The primary outcome measure was reported pain intensity provoked on 13 anatomical landmarks of the intra-pelvic side-wall. All women reported their perceived pain intensity for each anatomical landmark on Likert scales and an individual sum score was calculated. RESULTS: Women with chronic pelvic pain were older than women without CPP. At several intra-pelvic landmarks high intensity pain was provoked in women with CPP compared with less intense pain provoked at fewer landmarks in women without low back or pelvic pain (p < 0.0001). The average sum of pain intensity scores was about 4 times higher in women with CPP (1.3) as compared with those without low back or pelvic pain (0.3), p < 0.0001. This association remained when adjusting for the age difference between the pain groups in linear regression analysis. In addition, reported pain intensity at worst past week was independently associated with sum of pain intensity scores. The maximum individual sum of pain intensity scores among women without CPP was exceeded by that of 85% of the women with CPP. CONCLUSIONS: Parous women with CPP after childbirth had a heightened pain intensity over 13 anatomical landmarks during pelvic examination compared with parous women without CPP. These results need to be confirmed in a larger cohort with different types of CPP.

A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas.

Half of all human cancers lose p53 function by missense mutations, with an unknown fraction of these containing p53 in a self-aggregated amyloid-like state. Here we show that a cell-penetrating peptide, ReACp53, designed to inhibit p53 amyloid formation, rescues p53 function in cancer cell lines and in organoids derived from high-grade serous ovarian carcinomas (HGSOC), an aggressive cancer characterized by ubiquitous p53 mutations. Rescued p53 behaves similarly to its wild-type counterpart in regulating target genes, reducing cell proliferation and increasing cell death. Intraperitoneal administration decreases tumor proliferation and shrinks xenografts in vivo. Our data show the effectiveness of targeting a specific aggregation defect of p53 and its potential applicability to HGSOCs.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1.

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells.

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal. FINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression. CONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

Referred pain patterns provoked on intra-pelvic structures among women with and without chronic pelvic pain: a descriptive study.

OBJECTIVES: To describe referred pain patterns provoked from intra-pelvic structures in women with chronic pelvic pain (CPP) persisting after childbirth with the purpose to improve diagnostics and give implications for treatment. MATERIALS AND METHODS: In this descriptive and comparative study 36 parous women with CPP were recruited from a physiotherapy department waiting list and by advertisements in newspapers. A control group of 29 parous women without CPP was consecutively assessed for eligibility from a midwifery surgery. Inclusion criterion for CPP was: moderate pain in the sacral region persisting at least six months after childbirth confirmed by pelvic pain provocation tests. Exclusion criteria in groups with and without CPP were: persistent back or pelvic pain with onset prior to pregnancy, previous back surgery and positive neurological signs. Pain was provoked by palpation of 13 predetermined intra-pelvic anatomical landmarks. The referred pain distribution was expressed in pain drawings and described in pain maps and calculated referred pain areas. RESULTS: Pain provoked by palpation of the posterior intra-pelvic landmarks was mostly referred to the sacral region and pain provoked by palpation of the ischial and pubic bones was mostly referred to the groin and pubic regions, with or without pain referred down the ipsilateral leg. The average pain distribution area provoked by palpation of all 13 anatomical landmarks was 30.3 mm² (19.2 to 53.7) in women with CPP as compared to 3.2 mm² (1.0 to 5.1) in women without CPP, p< 0.0001. CONCLUSIONS: Referred pain patterns provoked from intra-pelvic landmarks in women with CPP are consistent with sclerotomal sensory innervation. Magnification of referred pain patterns indicates allodynia and central sensitization. The results suggest that pain mapping can be used to evaluate and confirm the pain experience among women with CPP and contribute to diagnosis.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

Finger joint laxity, number of previous pregnancies and pregnancy induced back pain in a cohort study.

BACKGROUND: General joint hypermobility is estimated to affect about 10% of the population and is a prerequisite of heritable connective tissue disorders where fragile connective tissue is a prominent feature. Pregnancy induced back pain is common whereas about 10% of women still have disabling pain several years after childbirth. The pathogenesis of the pain condition is uncertain, although several risk factors are suggested including general joint hypermobility. In the present study, the possible association of peripheral joint mobility in early pregnancy on the incidence of back pain with onset during pregnancy and persisting after childbirth was explored. METHODS: A cohort of 200 pregnant women recruited from antenatal health care clinics was assessed by questionnaire and clinical examination, including measurement of passive abduction of the left fourth finger, throughout pregnancy and at 13 weeks postpartum. Comparisons were made between women with and without back pain. Statistical tests used were χ2-test, t-test, Spearman correlation and multiple logistic regression. RESULTS: In the cohort, the mean passive abduction angle of the left fourth finger increased from 40.1° in early pregnancy to 41.8° at the postpartum appointment. At the postpartum appointment, women in the back pain group had a significantly larger mean passive abduction angle of the left fourth finger of 4.4°, twice as many previous pregnancies and deliveries, and more than twice as frequent back pain in previous pregnancy, as compared with women with no persistent back pain. A similar pattern was displayed in late pregnancy. In a multiple regression analysis, the passive abduction angle of the left fourth finger in early pregnancy and the number of previous pregnancies were positively, significantly and independently associated to the incidence of back pain in late pregnancy and postpartum. CONCLUSIONS: Finger joint laxity as a reflection of constitutional weakness of connective tissue and number of previous pregnancies were associated with the development of back pain induced in pregnancy and persisting after childbirth. These factors may provide a foundation for development of targeted prevention strategies, but this have to be confirmed in future research including measurement of general joint laxity.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Improved function in women with persistent pregnancy-related pelvic pain after a single corticosteroid injection to the ischiadic spine: a randomized double-blind controlled trial.

BACKGROUND: Pregnancy-related low back and pelvic pain is a worldwide problem. A large proportion of women still experience disabling daily back pain 2 years after childbirth, resulting in major changes in activities and general well-being. In spite of this, the source of pain and effective treatment are uncertain. OBJECTIVE: To evaluate the short-term effects on function of a single corticosteroid injection treatment to the ischiadic spine in women with persistent pregnancy-related pelvic pain (PPPP). METHODS: Thirty-six women were allocated to injection treatment with slow-release triamcinolone and lidocain or saline and lidocain, given once at the sacrospinous ligament insertion on the ischiadic spine bilaterally with follow-up at 4 weeks. Outcome measures were Disability Rating Index (DRI), self-rated functional health (SF-36), gait speed and endurance (6MWT), and strength and endurance of trunk muscles (isometric trunk extensor and flexor tests). RESULTS: Women in the triamcinolone group showed significantly improved DRI (p = 0.046), 6MWT (p = 0.016), and isometric trunk extensor tests (p = 0.004), as compared with the saline group. Close co-variation was shown between improved function and reduced pain intensity. CONCLUSIONS: Improved function was achieved among women with PPPP after a single injection treatment with slow-release corticosteroid. The effect was positively correlated to the reduced pain intensity.

High-throughput screening for bioactive molecules using primary cell culture of transgenic zebrafish embryos.

Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

Hydrodynamic stretching of single cells for large population mechanical phenotyping.

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Cdk2ap2 is a novel regulator for self-renewal of murine embryonic stem cells.

In this study we present data to support the role for Cdk2ap2 in regulating self-renewal of mouse embryonic stem cells (mESCs) under permissive conditions, and cell survival during differentiation of the mESCs into terminally differentiated cell types. To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2(tr/tr)). The Cdk2ap2(tr/tr) mESCs, when grown in a complete growth medium containing leukemia inhibitory factor (LIF), showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation, including Brachyury (T), Afp, and S100a, when compared to Wt mESCs. Cdk2ap2(tr/tr) mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Furthermore, Cdk2ap2(tr/tr) mESCs were unable to form teratomas in severe combined immunodeficiency (SCID) mice. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis.

Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells.

Pluripotent stem cells hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into any cell type of the body. In this study, we demonstrate that proper mitochondrial function is essential for proliferation of undifferentiated ESCs. Attenuating mitochondrial function under self-renewing conditions makes these cells more glycolytic-dependent, and it is associated with an increase in the mRNA reserves of Nanog, Oct4, and Sox2. In contrast, attenuating mitochondrial function during the first 7 days of differentiation results in normal repression of Oct4, Nanog, and Sox2. However, differentiation potential is compromised as revealed by abnormal transcription of multiple Hox genes. Furthermore, under differentiating conditions in which mitochondrial function is attenuated, tumorigenic cells continue to persist. Our results, therefore establish the importance of normal mitochondrial function in ESC proliferation, regulating differentiation, and preventing the emergence of tumorigenic cells during the process of differentiation.

Loss of Pten causes tumor initiation following differentiation of murine pluripotent stem cells due to failed repression of Nanog.

Pluripotent stem cells (PSCs) hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into every cell type in the body. One major barrier to the use of PSCs is their potential risk for tumor initiation following differentiation and transplantation in vivo. In the current study we sought to evaluate the role of the tumor suppressor Pten in murine PSC neoplastic progression. Using eight functional assays that have previously been used to indicate PSC adaptation or transformation, Pten null embryonic stem cells (ESCs) failed to rate as significant in five of them. Instead, our data demonstrate that the loss of Pten causes the emergence of a small number of aggressive, teratoma-initiating embryonic carcinoma cells (ECCs) during differentiation in vitro, while the remaining 90-95% of differentiated cells are non-tumorigenic. Furthermore, our data show that the mechanism by which Pten null ECCs emerge in vitro and cause tumors in vivo is through increased survival and self-renewal, due to failed repression of the transcription factor Nanog.

Female human iPSCs retain an inactive X chromosome.

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), as in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X inactivation with two active Xs as found in some female human embryonic stem cell lines. Furthermore, whereas fibroblasts are mosaic for the Xi, hiPSCs are clonal. This nonrandom pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.