Cord-stromal Cell Research Articles

Wharton\u2019s jelly mesenchymal stem cell-based or umbilical vein endothelial cell-based serum-free coculture with cytokines supports the ex vivo expansion/maintenance of cord blood hematopoietic stem/progenitor cells

BackgroundThe umbilical cord blood (UCB) has been widely accepted as an alternative source of hematopoietic stem/progenitor cells (HSPCs) for transplantation, and its use in adults is still restricted because of low absolute numbers. To overcome this obstacle, expansion of UCB-HSPCs under feeder cell-based coculture is a promising possibility. In this study, we explored UCB-CD34+ cells ex vivo expansion using Wharton’s jelly mesenchymal stem cells (WJ-MSCs) or umbilical vein endothelial cells (UVECs) as feeder layer-based serum-free coculture system with a cocktail of cytokines.MethodsUCB-CD34+ cells were cultured in five different coculture conditions composed of umbilical cord stromal cells (WJ-MSCs or UVECs) with or without a cocktail of cytokines (SCF, FLT3L, and TPO). The cultured cells were harvested at day 10 and analyzed for phenotypes and functionalities, including total nuclear cells (TNCs), CD34+ cells, CD34+CD38− cells, colony-forming unit (CFU) for committed progenitors, and long-term culture initiating cells (LTC-ICs) for HSPCs.ResultsOur work showed the numbers of TNC cells, CD34+ cells, and CD34+CD38− cells were expanded under five coculture conditions, and the feeder layer-based cocultures further promoted the expansion. The numbers of colonies of CFU-GM, CFU-E/BFU-E, and CFU-GEMM in the cocultures with cytokines were significantly higher than their counterparts at day 0 (p < 0.05), while no significant difference (p > 0.05) in those without the addition of cytokines. The numbers of LTC-ICs were increased both under the WJ-MSCs and UVECs with cytokine cocultures, but only in the UVECs group showed a significant difference (p < 0.05), and were decreased under conditions without cytokine (p < 0.05).ConclusionOur data demonstrate that both WJ-MSCs and UVECs as feeder layer could efficiently support the expansion of UCB-CD34+ cells in synergy with SCF, FLT3L, and TPO under serum-free culture condition. The UVECs combined with the 3GF cytokine cocktail could maintain the growth of LTC-ICs derived from UCB-CD34+ cells and even expand to some extent.

Роль суммарных РНК лимфоидных и стволовых клеток в коррекции уровня глюкозы в крови при экспериментальном сахарном диабете

Цель - оценка влияния препаратов суммарной РНК лимфоидных клеток на уровень глюкозы в крови при экспериментальном аллоксановом диабете. Методика. Работа выполнена на 68 белых беспородных крысах обоего пола массой 180-220 г. Для моделирования аллоксанового сахарного диабета 1 типа животным однократно подкожно вводили полный адъювант Фрейнда в дозе 0,5 мл/крысу. Через 24 ч (на фоне 24-часового голодания, при свободном доступе к воде) однократно подкожно вводили препарат аллоксан тригидрат («La Chema», Чехия) в дозе 200 мг/кг (4% раствор в 0,9% NaCl). Для предотвращения фатального кетоацидоза, начиная с 3-х сут после введения аллоксана, все крысы получали базисную инсулинотерапию. В экспериментах использовано 8 разновидностей препаратов суммарной РНК: из лимфоидных клеток селезенки, из костного мозга и хвостовой части поджелудочной железы интактных крыс (РНКс-1, РНКкм-1 и РНКп/ж, соответственно); из лимфоидных клеток селезенки крыс, подвергнутых кровопусканию в объеме 2% от массы тела за 17 ч до выделения РНК (РНКс-2 с индуцированной повышенной морфогенетической активностью); из лимфоидных клеток селезенки и тимуса крыс, подвергнутых кровопусканию в объеме 2% от массы тела за 96 ч до выделения РНК (РНКс-3 и РНКт-3, соответственно, с высокой ингибирующей морфогенез активностью); из лимфоцитов периферической крови здорового человека (РНКлпкч), а также из стволовых клеток стромы пупочного канатика человека. Результаты. Показана возможность восстановления функции инсулярного аппарата и стойкой нормализации уровня глюкозы в крови крыс с экспериментальным аллоксановым диабетом 1 типа. Установлено наличие корригирующей способности аллогенных и ксеногенных препаратов суммарной РНК, выделенной из лимфоидных органов крыс и лимфоцитов периферической крови человека, а также из мезенхимных стромальных клеток пуповины человека, в отношении уровня глюкозы крови у крыс. Продемонстрирован различный вклад препаратов суммарной РНК, полученной из разных лимфоидных органов и стволовых клеток, в восстановлении нормального уровня глюкозы в крови животных. Установлено, что указанные препараты действуют на разные ткани-мишени в процессе восстановления функции инсулярного аппарата. Заключение. Полученные данные свидетельствуют о принципиальной возможности эффективного лечения сахарного диабета 1 и 2 типов. Доказана эффективность неинвазивного интраназального введения аллогенных и ксеногенных препаратов суммарной РНК лимфоидных и стволовых клеток. Полученные результаты ставят вопрос о необходимости разработки более адекватной экспериментальной модели, а также о перспективности поиска подходов к персонифицированному индивидуальному лечению этой патологии с учетом особенностей ее развития в каждом конкретном случае. Aim. To study the effect of total RNA from lymphoid cells on blood glucose in experimental alloxan diabetes mellitus (DM). Methods. The study was conducted on 68 white mongrel rats of both sexes weighing 180-220 g. Alloxan DM was modeled with full Freund’s adjuvant (0.5 ml/rat). After 24 h of fasting with free access to water, a single dose of alloxan trihydrate (La Chemas, Czeck Republic) was injected subcutaneously (200 mg/kg as a 4% solution in saline). All rats received basis insulin therapy starting from day 3 of the alloxan injection to prevent fatal ketoacidosis. Eight types of total RNA were used: from splenic lymphoid cells, bone marrow, and caudal part of the pancreas of intact rats; from splenic lymphoid cells of rats after blood withdrawal (2% of body weight 17 h prior to RNA isolation) (RNA after induction of increased morphogenetic activity); from splenic and thymic lymphoid cells of rats after blood withdrawal (2% of body weight 96 h prior to RNA isolation) (RNAs with high morphogenesis-inhibiting activity); and from peripheral blood lymphocytes and umbilical cord stromal stem cells of a healthy human. Results. The study showed a possibility for functional recovery of the insular apparatus and stable normalization of blood glucose level in rats with experimental alloxan DM, a model of clinical type 1 DM. Allogenic and xenogeneic total RNAs isolated from rat lymphoid organs, peripheral blood lymphocytes, and mesenchymal stromal cells of human umbilical cord were able to correct the blood glucose level in rats. Total RNAs isolated from different lymphoid organs and stem cells differently contributed to normalization of blood glucose in rats. These total RNAs were shown to influence different target tissues during restoration of the insular apparatus function. Conclusion. The study showed a principle possibility of effective therapy for types 1 and 2 DM and demonstrated the efficacy of non-invasive intranasal administration of allogenic and xenogeneic total RNAs from lymphoid and stem cells. These results indicated a need for developing a more relevant experimental model and offered a promising outlook for individualized treatment of this disease with due account of its peculiar features in each specific case. The study was conducted as a part of the Program for Basic Research of State Academies of Science in 2013-2020 on Development of Cell Models for Molecular Processes in Organs and Tissue (V.N. Orekhovich Research Institute of Biomedical Chemistry) and the complex Research Work #01201354257 on Regulation of Hemopoietic and Nonhemopoietic Functions of Bone Marrow Cells in Experiment and Clinic (Sounth Ural State Medical University).

SAT-289 An Unusual Virilizing Ovarian Tumor in Adolescence

Background: The etiology of hyperandrogenism in adolescent females includes polycystic ovarian syndrome, non-classical congenital adrenal hyperplasia (CAH), Cushing’s syndrome, androgen-secreting tumors (adrenal or ovarian) or exogenous use. Ovarian tumors producing androgens comprise <5% of all ovarian neoplasms and usually originate from sex cord stromal cells. There are sporadic case reports of mature cystic teratomas (MCTs), a type of germ cell tumor, causing hyperandrogenism in women but only two reported cases of virilizing MCTs in adolescent girls (1, 2). We present a third case of this nature. Case presentation: A 17-year old girl with no past medical history presented with worsening abdominal distension and discomfort. She was also noted to have excessive thick facial hair which she stated had been present throughout adolescence. She reported recent irregularity in her menstrual periods. She had no history genital abnormalities at birth or premature adrenarche, thelarche or menarche. She denied using anabolic steroids or other medications. Family history was negative for CAH or sudden infant death. On examination, there was significant abdominal distension, terminal hair over the chin, upper lip, side-burns and abdomen, as well as severe clitoromegaly with prominent glans measuring 3cm x 1.5cm, but no evidence of acanthosis nigricans. Chemistry showed testosterone of 178.3 ng/dL (3.75 - 49.57 ng/dL), DHEA-S of 550.4 mcg/dL (150.0 - 590.0 mcg/dL) and 17-hydroxyprogesterone of 224.7 ng/dL (26 - 325 ng/dL). Her abdomino-pelvic ultrasound and CT scan showed a 35.5 cm cystic mass with focal fatty components and dystrophic calcifications suggestive of MCT. Adrenal glands were normal. The patient underwent an exploratory laparotomy with left salpingo-oopherectomy and right cystectomy. Preliminary pathology results showed bilateral MCTs. Two weeks post-operatively, testosterone level decreased to 32.5 ng/dL. Conclusion: Given the normalization of testosterone after surgery, the bilateral MCTs are the suspected source of the patient’s androgen excess. MCTs are the most common ovarian neoplasm in adolescent females, but they are an exceedingly rare cause of hyperandrogenemia. They can be slow-growing and local manifestations may not present until years later. However, testosterone-secreting MCTs may be brought to a physician’s attention due to signs of virilization. A multidisciplinary approach with primary care physicians, endocrinologists, gynecologists and pathologists is needed to make an accurate diagnosis in a timely manner.

PARAGON: A phase 2 study of anastrozole (An) in patients with estrogen receptor(ER) and / progesterone receptor (PR) positive recurrent/metastatic granulosa cell tumors/sex-cord stromal tumors (GCT) of the ovary.

5524Background: GCTs occur predominantly in postmenopausal women and arise from sex cord stromal cells of the ovary. They generally have a good prognosis but can metastasize / recur after surgery. ...

Pure leydig cell tumour – a rare virilizing tumour in a young female

Sex cord stromal cell tumours constitute 5-8% of all ovarian neoplasms of which pure leydig cell tumours constitute 0.1%. These tumours are most commonly found in the post-menopausal age group and patients present with a rapid onset of symptoms of androgen excess like hoarseness of voice, clitoromegaly and hirsutism. We present a case of 39 year old female, who presented with virilising symptoms since 2 years. Serum Testosterone levels were raised and CT revealed homogeneously enhancing mass of 2.8 cm in size in the right ovary. An unilateral oophorectomy was done and sent for histopathological examination. On Gross examination, the ovary revealed the presence of a well circumscribed greyish white tumour with multiple yellowish areas. Microscopy revealed features suggestive of pure leydig cell tumour with reinke crystals. Post operatively, the patient improved symptomatically. The unusual features seen in this case were younger age at presentation and insidious onset of symptoms. The presentation of pure leydig cell tumour- hilar type at such a young age is extremely uncommon.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation.Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC.Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells.Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

Steroid cell tumour- NOS of the ovary in a young female

Sex cord stromal cell tumours constitute 5-8 % of all ovarian neoplasms. Steroid cell tumours are a type of sex cord stromal cell tumours. The name “Steroid cell” stands for the morphology and the functionality of these tumours. Steroid cell tumours- Not otherwise specified constitute about 56% of all steroid cell tumours and presents more commonly with androgenic manifestations in third to fourth decade. We present a case, 22 year old, who presented with virilizing symptoms. MRI was suggestive of an right ovarian mass for which right salpingo-oophorectomy was done. Histopathology revealed features classical of Steroid cell tumour- Not otherwise specified. We present the case for a relatively younger age at presentation and the classical histomorphology. DOI:10.21276/APALM.1597

Umbilical cord stromal cell (UCSC) exosomes protect tubular epithelial cells from gentamicin injury

Bone Marrow Mesenchymal Stromal Cells (MSCs) and Umbilical Cord Stromal Cells (UCSC) protect the kidney against nephrotoxic insults. This effect could be paracrine, possibly mediated by extracellular vesicles. Exosomes (EXOs) are extracellular vesicles that transfer mRNA and microRNA to the recipient cells, mediating the communication between them. Mir 126 mediates the protective effect of stem cell microvesicles. The goal of the present study was to analyze the effects of EXOs from MSCs and UCSC on proximal tubular epithelial cells from rats (RPTC) exposed to gentamicin (genta). UCSC and MSCs were maintained in culture conditions and every 3 days the culture medium was collected. EXOs were extracted through filtration in the 0.1μm filter, followed by ultracentrifugation (100,000g during 2h). Mir 126 from stromal cells and EXOs were extracted and analyzed by real time‐PCR. RPTC cells were exposed to gentamicin (2mM) in the presence or absence of exosomes (50 μg/ml), DNA damage and proliferation was analyzed by H2Ax and BrDU immunofluorescence, respectively. The expression of p21 and c‐myc was analyzed in RPTC through western blot. Mir 126 expression was increased in exosomes in comparison to stromal cells. It was increased in BM‐MSC exosomes (2.52 ± 0.12 AU) in comparison to UCSC exosomes (1.2 ± 0.2 AU). The treatment with UCSC‐EXO, but not with MSC‐EXO, stimulated the proliferation and decreased DNA damage induced by gentamicin in tubular cells. MSC‐EXO increased proliferation and decreased DNA damage when mir126 was depleted from exosomes with 126 antimir. RPTC cells treated with UCSC‐EXO increased the tumor suppressor protein p21 and increased in cell cycle progress protein c‐myc. MSC‐EXO did not show the same effect. Our results suggest that stromal cell exosomes carry mir 126 and this microRNA could be related to the paracrine protective effect of umbilical stromal cell exosome.Support or Funding InformationSupported by FAPESP, CNPq, CAPES and FOR

Histological Study of Bone Marrow and Umbilical Cord Stromal Cell Transplantation in Regenerating Rat Peripheral Nerve.

Bone marrow and umbilical cord stromal cells are multipotential stem cells that have the ability to produce growth factors that play an important role in survival and generation of axons. The goal of this study was to evaluate the effects of the two different mesenchymal stem cells on peripheral nerve regeneration. In this experimental study, a 10 mm segment of the left sciatic nerve of male Wistar rats (250-300 g) was removed with a silicone tube interposed into this nerve gap. Bone marrow stromal cells (BMSCs) and human umbilical cord stromal cells (HUCSCs) were respectively obtained from rat and human. The cells were sepa- rately cultured and transplanted into the nerve gap. The sciatic nerve regeneration was evaluated by immunohistochemistry, and light and electron microscopy. Moreover, histo- morphology of the gastrocnemius muscle was observed. The nerve regeneration in the BMSCs and HUCSCs groups that had received the stem cells was significantly more favorable than the control group. In addition, the BM- SCs group was significantly more favorable than the HUCSCs group (P<0.05). The results of this study suggest that both homograft BMSCs and het- erograft HUCSCs may have the potential to regenerate peripheral nerve injury and transplantation of BMSCs may be more effective than HUCSCs in rat.

Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway

Trophic factors (TFs) play important role during development and adult tissue maintenance. In neurodegenerative diseases (ND) TF supplementation provides protection. Stromal cells (HUMS) derived from the human umbilical cord matrix provide neuroprotection in the ND models of mice. Though TF mediated protection is known, the exact mechanism of protection is not clear. So, here the essential TFs (secreted by HUMS cells) and the pathway of induction of neurite extension, differentiation and networking is addressed. The HUMS cells from the human umbilical cord matrix were derived and the mouse spinal cord motor neuron cell line, NSC-34 was extensively used. Flow cytometry, immunohistochemistry, RT- PCR, western blot, ELISA and antibody/inhibitor treatment were carried out to figure out the TF pathway. The HUMS cells secrete six neurotrophic factors (sTFs), namely, NT-3, NGF, BDNF, VEGF, IGF-1 and GDNF (TFs). These TFs are sufficient to induce differentiation, neurite extension and neural networking in a motor neuron cell line, NSC34. All the 5 TFs need to be neutralized simultaneously with their antibodies to abrogate neurite extension. These motor neurons express the concomitant receptors, which are either receptor tyrosine kinase (TrK) coupled or to the receptor followed by the TrKs, for the above trophic factors (except for BDNF). The tyrosine kinase inhibitor, K252a, drastically reduces neurite extension. In NSC34, the TFs are coupled to the PI3K-Akt-pathway and the RAS-MAP kinase signaling through phosphorylation of ERK1 and ERK2. PI3K inhibitor, Ly 294002, abolishes neural differentiation and neurite extension. Thus, differentiation, neurite extension and networking could be achieved through the PI3K pathway. Intriguingly, the cAMP second messenger system coupling was not required. H89, PKA-inhibitor caused extensive cell death. But, had no effect in the presence of HUMS-secreted-TFs(HSTFs) suggesting a pathway switch for cell survival itself. HUMS cells and their secreted factors could be of great use in regenerative medicine (RM). The activators of PI3K pathway, the major route of these HUMS-TFs action could be explored in RM and in the neurobiology of neural differentiation and extension.

Clinical neurorestorative progress in traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of death and disability from trauma to the central nervous system. Besides the surgical interventions and symptomatic management, the conventional therapies for TBI and its sequelae are still limited. Recently emerging evidence suggests that some neurorestorative treatments appear to have a potential therapeutic role for TBI and improving the patient's quality of life. The current clinical neurorestorative strategies available in TBI include pharmacological treatments (recombinant human interleukin-1 receptor antagonist, amantadine, lithium, and valproate), the neuromodulation treatments (repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and low-level laser therapy), cell transplantation (bone marrow stromal cells and umbilical cord stromal cells), and combined neurorehabilitation. In this review, we summarize the recent clinical neurorestorative progress in the management of neurodegeneration as well as cognitive and motor deficits after TBI; indeed further clinical trials are required to provide more robust evidence.

Anticancer properties of peptide fragments of hair proteins.

The primary function of hair and fur covering mammalian skin is to provide mechanical and thermal protection for the body. The proteins that constitute hair are extremely resistant to degradation by environmental factors. However, even durable materials can be slowly broken down by mechanical stresses, biodegradation mediated by endogenous enzymes in the skin or host microbes. We hypothesised that the biodegradation products of hair may possess bioprotective properties, which supplement their physical protective properties. Although evolutionary processes have led to a reduction in the amount of hair on the human body, it is possible that the bioprotective properties of hair biodegradation products have persisted. The human skin is exposed to various environmental carcinogenic factors. Therefore, we hypothesised that the potential bioprotective mechanisms of hair degradation products affect melanoma growth. We used pepsin to partially digest hair enzymatically, and this process produced a water-soluble lysate containing a mixture of peptides, including fragments of keratin and keratin-associated proteins. We found out that the mixtures of soluble peptides obtained from human hair inhibited the proliferation of human melanoma cells in vitro. Moreover, the hair-derived peptide mixtures also inhibited the proliferation of B lymphoma cells and urinary bladder cancer cells. Normal human cells varied in their susceptibility to the effects of the lysate; the hair-derived peptide mixtures modulated the proliferation of normal human fibroblasts but did not inhibit the proliferation of human mesenchymal cells derived from umbilical cord stromal cells. These results suggest that hair-derived peptides may represent a new class of anti-proliferative factors derived from basically structural proteins. Identification of active regulatory compounds and recognition of the mechanism of their action might pave the way to elaboration of new anticancer drugs.

Les tumeurs de la granulosa de l’ovaire

Ovarian granulosa cell tumors (TGO) are rare neoplasms. They arise from sex cord stromal cells of the ovaries. They are characterized by their slow natural history, and their tendency to relapse long time after the initial diagnosis. Complete staging surgery of the disease is the cornerstone of treatment. Chemotherapy is indicated for localized tumors with a high risk of recurrence, and for recurrent or advanced tumors. Prolonged follow-up is recommended.

Genetic changes in nonepithelial ovarian cancer

Nonepithelial ovarian cancers (OCs), including sex cord-stromal tumors (SCSTs) and germ cell tumors (GCTs), are an uncommon subset of OC, together accounting for 10% of all OCs. The etiology of these tumors remains largely unresolved. It is well established that tumorigenesis is the result of multiple genetic alterations driving a normal cell toward a malignant state. Much effort has been made into researching the molecular mechanisms underlying epithelial OC, but far less is known about the genetic changes in SCSTs and GCTs. Recently, a single point missense mutation (C134W) was found in the FOXL2 gene in approximately 95% of adult-type granulosa cell tumors, suggesting a key role for FOXL2 in these tumors. By contrast, the FOXL2 mutation was not found in the juvenile type. DICER1 somatic missense mutations were found in approximately 60% of Sertoli-Leydig tumors. Ovarian GCTs share many morphological features and a similar pattern of chromosomal alterations with testicular GCTs. In the latter, recent genome-wide association studies have identified seven susceptibility loci near KITLG, SPRY4, UKC2, BAK1, DMRT1, TERT and ATF7IP. All of the susceptibility loci detected thus far are all involved in primordial germ cell function or sex determination. TGF-β/BMP and Wnt/β-catenin signaling was absent in dysgerminomas, but present in yolk sac tumors, suggesting intertumoral heterogeneity. In this article, the authors aim to provide an overview of the current knowledge on the possible molecular changes in SCSTs and GCTs of the ovary.

Expression of SALL4 and SF-1 in Gonadoblastoma

Gonadoblastoma is a rare gonadal neoplasm composed of primordial germ cells and sex cord-stromal cells and usually occurs in patients with dysgenetic gonads. When patients with gonadoblastoma develop an invasive germ cell tumor, the invasive germ cell component can take the form of dysgerminoma/seminoma, embryonal carcinoma, or yolk sac tumor. In this study, we performed immunohistochemical analysis for SALL4 and steroidogenic factor-1 (SF-1) on 4 cases of gonadoblastoma to examine the expression patterns of these proteins. All of the patients were phenotypically female. One patient had Swyer syndrome, the rest had Turner syndrome. The primordial germ cell component was histologically similar to cells in dysgerminoma/seminoma in these 4 cases. Two patients showed the invasive component-dysgerminoma. As expected, SALL4 stained the germ cells and SF-1 stained the sex cord-stromal cells. There was a clear distinction between the staining patterns of these 2 cell populations. This study demonstrates the utility of SALL4 and SF-1 in determining whether or not there is an invasion in the primordial germ cell component.

The Diagnostic Utility of Intraoperative Cytology in the Management of Ovarian Tumours

The present study was done to evaluate the status of intraoperative cytology as a diagnostic and supportive investigation for ovarian tumours. The present study was conducted in the Department of Pathology, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India during a time span of 13 months (June 2011 to June 2012). A prospective investigation was performed on 50 cases of suspected ovarian neoplasms. Imprint smears were made intraoperatively from fresh samples from various representative areas, and they were immediately fixed in 95% ethyl alcohol and stained with the haematoxylin and eosin stain. The results were compared with the final histopathological diagnoses in each case. There were 20 benign lesions, 4 borderline epithelial neoplasms and 26 malignant tumours according to the final histopathological diagnosis. The overall diagnostic accuracy of imprint cytology has been satisfactory, with those of 90% of the cases correlating with the final diagnoses. Characteristic cytological patterns were noted in various surface epithelial, sex cord stromal and germ cell tumours. Imprint cytology can be used as an adjunct to histopathology for a rapid and an early diagnosis in the operation theatre, particularly in developing countries like ours, where the facility of frozen sections is often not available, since a rapid preliminary diagnosis may help in the surgical management planning.

Granulosa Cells in the Uterosacral Ligament: Case Report and Review of the Literature

Granulosa cells are components of the sex cord–stromal cells in the ovary responsible for steroidogenesis. Uncommonly, extraovarian granulosa cells have been reported to be associated with malignant processes of the ovary. We report a unique case of benign granulosa cells, found during routine laparoscopic evaluation, in the uterosacral ligaments in a 20-year-old patient with chronic pelvic pain and infertility. Possible mechanisms include implantation of released granulosa cells from a normal ovary or arising from a focus of müllerianosis. Of note, a focus of endosalpingiosis and endometriosis was also identified within the specimen.

Umbilical Cord Stromal Cell Differentiation: little correlation with known markers of the chondrocyte phenotype or cartilage extracellular matrix

The increasing prevalence of degenerative joint diseases, in particular osteoarthritis, has driven the quest for chondrocyte precursor cells and biomaterials suitable for autologous cartilage repair (ACR). As an alternative to mesenchymal stem cells (MSCs) generated in situ from the subchondral bone, the differentiation and expansion of autologous MSCs in the presence of pro-chondrogenic factors such as BMP-2 and/or TGF beta holds great promise as a therapeutic approach. However, a typical feature of these chondrogenesis systems, including MSCs derived from umbilical cord blood (1) and the stromal model used in the current report (2) is differentiation towards hypertrophy marked by the production of collagen X. To quote Hardingham et al., (3) “chondrocyte hypertrophy and ECM calcification would be an undesirable characteristic of any cartilage repair tissue.” Central to the study by De la Fuente et al., (4) is the claim that umbilical cord MSCs are “similar to chondrocytes and expressed proteins characteristic of the cartilage extracellular matrix”. Apart from the immunohistochemistry for collagen II published previously (2), there is scant evidence to support this claim, as none of the proteomic data provides specific evidence for biosynthesis of a cartilaginous matrix. The cartilage ECM contains a plethora of well-known proteoglycans, non- collagenous proteins and glycoproteins. None of the typical cartilage ECM components, including aggrecan, link protein and the matrilins are found in this study and neither are the sensitive markers for the chondrocyte differentiation status such as cartilage oligomeric matrix protein and collagen IX (5). The authors instead report a collection of proteins (PLOD2, PDI, GRP78, PPIA and HSP90) that are involved in protein biosynthesis and post- translational modification and are found in a diverse range of tissues, if not ubiquitous. The claim that these proteins are involved in “cartilage extracellular matrix metabolism” is misleading, as none of these proteins are cartilage-specific. A second set of differentially- abundant proteins (CALU, CALR3, VIM, PDIA3, ZYX and ANXA5 in Table III), was provided as evidence of chondrogenic differentiation: “Results demonstrate that MSCs cultured in chondrogenic media become chondrocyte- like, as the cells express proteins observed in differentiated chondrocytes or in cartilage”. Again, these proteins are expressed in bone cells (eg osteoblasts), fibroblasts etc, and so their altered expression reveals nothing about the differentiation of the MSCs into “chondrocyte-like” cells. None are chondrocyte-specific. Finally, these results are drawn together in a protein network diagram summarizing the relationships between the proteins based on “linked bibliographic information”. It is difficult to extract any biological significance from this figure without all the citations on which these connections are based, but many of the connections are tenuous and some are contradictory. For example, the intermediate filament protein vimentin, linked to “extracellular matrix proteins” and “cartilage”, is down regulated in the current report. However this contradicts the findings of Bobick et al., (6) showing a positive role for vimentin in chondrogenesis. The field of autogolous cartilage repair has been plagued by the well documented phenotypic instability of chondrocytes: de-differentiation towards fibroblastic cells and/or the uncontrolled hypertrophy and matrix calcification analogous to differentiation of growth plate chondrocytes. The holy grail of ACR is the suppression these processes (marked by expression of collagen I and collagen X, respectively) during the expansion of phenotypically stable MSC precursors. Given the critical role of the ECM in cartilage function, proteomics is a promising method for determining the optimal source of MSCs, as the matrix proteins accumulated at the time of sampling may not be reflected by their respective mRNA levels. I anticipate that further studies using proteomics to compare engineered constructs and authentic cartilage will lead to a greater understanding of MSC-based chondrogenesis and improved materials for tissue engineering.

Mesenchymal cell populations: development of the induction systems for Schwann cells and neuronal cells and finding the unique stem cell population

Mesenchymal cell populations, referred to as mesenchymal stem cells or multipotent stromal cells (MSCs), which include bone marrow stromal cells (BMSCs), umbilical cord stromal cells and adipose stromal cells (ASCs), participate in tissue repair when transplanted into damaged or degenerating tissues. The trophic support and immunomodulation provided by MSCs can protect against tissue damage, and the differentiation potential of these cells may help to replace lost cells. MSCs are easily accessible and can be expanded on a large scale. In addition, BMSCs and ASCs can be harvested from the patient himself. Thus, MSCs are considered promising candidates for cell therapy. In this review, I will discuss recently discovered high-efficiency induction systems for deriving Schwann cells and neurons from MSCs. Other features of MSCs that are important for tissue repair include the self-renewing property of stem cells and their potential for differentiation. Thus, I will also discuss the stemness of MSCs and describe the discovery of a certain stem cell type among adult MSCs that can self-renew and differentiate into cells of all three germ layers. Furthermore, I will explore the prospects of using this cell population for cell therapy.

FOXL2 and SOX9 Distinguish the Lineage of the Sex Cord–Stromal Cells in Gonadoblastomas

Gonadoblastomas are mixed germ cell sex cord-stromal tumors that arise in dysgenetic gonads and are composed of immature germ cells and sex cord-stromal cells of indeterminate differentiation. FOXL2 is one of the first genes expressed in female gonad development, and it is required for proper granulosa cell differentiation during folliculogenesis. SOX9 , a downstream target of SRY , the gene in the Y chromosomal sex-determining region, is required for testicular development and for the formation and maintenance of (pre-)Sertoli cells. This study characterized the sex cord-stromal cells of gonadoblastoma by evaluating the expression of these counteracting transcription factors. Archival paraffin-embedded material of 7 gonadoblastomas, 5 of which were overgrown by dysgerminoma, was examined by immunohistochemistry for expression and localization of FOXL2 and SOX9. The sex cord-stromal cells revealed strong nuclear staining for FOXL2 and were negative for SOX9 expression. Germ cells in the gonadoblastoma and dysgerminoma components showed no FOXL2 and SOX9 expression. Areas of transition between gonadoblastoma and dysgerminoma revealed nests with a gradual reduction of FOXL2 expression. Our results support the hypothesis that the sex cord-stromal cell component of gonadoblastomas is of granulosa cell origin. In addition, FOXL2 appears to be a useful marker for the evaluation of overgrowth by dysgerminomas and for the identification of the transition zone of "dysgerminoma in situ." As FOXL2 and SOX9 are differentially expressed, they also should be useful for distinguishing gonadoblastomas from intratubular germ cell neoplasias and can help to differentiate those with a Sertoli cell component from gonadoblastoma with a granulosa cell component.