Two-Layer Compared With One-Layer Vaginal Cuff Closure at the Time of Total Laparoscopic Hysterectomy to Reduce Complications.

OBJECTIVE: To explore whether two-layer laparoscopic vaginal cuff closure at the time of laparoscopic hysterectomy is associated with a lower rate of postoperative complications compared with a standard one-layer cuff closure. METHODS: A retrospective cohort study of total laparoscopic hysterectomies performed by fellowship-trained minimally invasive gynecologic surgeons between 2011 and 2017 was performed. Surgeons sutured the vaginal cuff laparoscopically, either in a two- or one-layer closure. The primary outcome was a composite of total postoperative complications, including all medical and surgical complications within 30 days and vaginal cuff complications within 180 days. Factors known to influence laparoscopic vaginal cuff complications including age, postmenopausal status, body mass index, tobacco use, and immunosuppressant medications were examined and controlled for, while surgeon skill, colpotomy technique, and suture material remained standardized. We conducted statistical analyses including χ2, Fisher exact test, logistic regression, and post hoc power calculations. RESULTS: Of the 2,973 women who underwent total laparoscopic hysterectomies, 40.8% (n=1,213) of vaginal cuffs were closed with a two-layer closure and 59.2% (n=1,760) with a one-layer technique. Two-layer vaginal cuff closure was associated with decreased numbers of total postoperative complications (3.5% vs 5.7%; P<.01). The primary difference stemmed from lower vaginal cuff complications within 180 days (0.9% vs 2.6%; P<.01); no differences in 30-day medical and surgical postoperative complications were observed between the two groups (2.6% vs 3.1%; P=.77). No patients in the two-layer vaginal cuff closure cohort experienced a vaginal cuff dehiscence or mucosal separation compared with 1.0% in the one-layer group (P<.01). Compared with a one-layer closure, a two-layer closure was protective from postoperative complications (adjusted odds ratio 0.38, 95% CI 0.19-0.74). CONCLUSION: Although postoperative complications with laparoscopic hysterectomies are rare, two-layer laparoscopic vaginal cuff closure is associated with lower total postoperative complications compared with a one-layer closure. The difference was primary driven by cuff complications.

Enhanced recovery after surgery outcomes in minimally invasive nonhysterectomy gynecologic procedures.

BACKGROUND: Improved patient outcomes and satisfaction associated with enhanced recovery after surgery protocols have increasingly replaced traditional perioperative anesthesia care. Fast-track surgery pathways have been extensively validated in patients undergoing hysterectomies, yet the impact on fertility-sparing laparoscopic gynecologic operations, particularly those addressing chronic pain conditions, has not been examined. OBJECTIVE: The objective of the study was to determine the effects of enhanced recovery after surgery pathway implementation compared with conventional perioperative care in women undergoing laparoscopic minimally invasive nonhysterectomy gynecologic procedures. STUDY DESIGN: We conducted a retrospective cohort study of women undergoing uterine-sparing laparoscopic gynecologic procedures for benign conditions (tubal/adnexal pathology, endometriosis, or leiomyomas) during a 24 month period before and after enhanced recovery after surgery implementation at a tertiary care center. We compared immediate perioperative outcomes and 30 day complications. The primary outcome was same-day discharge rates. Factors influencing unplanned admissions, postoperative pain, sedation, nausea, and vomiting represented secondary analyses. RESULTS: A total of 410 women (enhanced recovery after surgery, n = 196; conventional perioperative care, n = 214) met inclusion criteria. Following enhanced recovery after surgery implementation, same-day discharge rates increased by 9.4% (P = .001). Reductions in postoperative pain and nausea/vomiting represented the primary driving factor behind lower unplanned admissions. Higher preoperative antiemetic medication administration in the enhanced recovery after surgery group resulted in a 57% reduction in postanesthesia care unit antiemetics (P < .001). Total perioperative narcotic medication use was also significantly reduced by 64% (P < .001), and the enhanced recovery after surgery cohort still demonstrated significantly lower postanesthesia unit care pain scores at hours 2 and 3 (P < .001). A 19 minute shorter postanesthesia care unit stay was noted in the enhanced recovery after surgery cohort (P = .036). Increased same-day discharge did not lead to higher postoperative complications or changes in 30 day emergency department visits or readmissions in patients with enhanced recovery after surgery. CONCLUSION: Enhanced recovery after surgery implementation resulted in increased same-day discharge rates and improved perioperative outcomes without affecting 30 day morbidity in women undergoing laparoscopic minimally invasive nonhysterectomy gynecologic procedures.

Tigers in the Sidewall: Surgical Approaches to Excision of Lateral Deep Infiltrating Endometriosis.

OBJECTIVE: To describe the surgical approaches and excisional techniques used in an extreme case of deep infiltrating endometriosis (DIE) affecting the lateral pelvic side wall. DESIGN: A technical video showing the excision of advanced lateral DIE. SETTING: An academic tertiary care hospital. INTERVENTIONS: A 32-year-old woman, gravida 2, para 1, presented for definitive surgical management of endometriosis-associated pelvic pain. Intraoperative findings revealed severe retroperitoneal fibrosis tethering the external iliac vein, internal iliac artery, obturator nerve, medial umbilical ligament, and ureter. The patient underwent laparoscopic management of the DIE involving the lateral pelvic side wall. We demonstrate the surgical methods and tools required to overcome a unique endometriotic nodule that would not allow for traditional lysis of adhesions from the pelvic side wall. Instead, we used a nontraditional surgical approach by tunneling under the external iliac vascular to tackle the dissection from a lateral to medial direction to free the obturator nerve and internal iliac artery from the ureter and endometriotic nodule. CONCLUSION: Extreme cases of DIE involving the pelvic side wall require surgical finesse when normal planes of dissection are obliterated. Knowledge of retroperitoneal anatomy is critical to overcome unexpected lateral pelvic side wall endometriosis because the disease is rarely confined to the surface. Innovative surgical thinking complemented by an array of surgical tools will ultimately allow the surgeon to master these difficult endometriotic resections.

The role of appendectomy at the time of laparoscopic surgery for benign gynecologic conditions.

PURPOSE OF REVIEW: The risk-benefit ratio of concurrent appendectomy at the time of gynecologic surgery has long been debated and remains controversial. However, emerging data on the appendix's role in chronic pain syndromes point to a previously unrecognized link between gynecologic disorders and appendicular pathology. In this article, we review the indications for appendectomy at the time of laparoscopic gynecologic surgery for the treatment of endometriosis and chronic pelvic pain. RECENT FINDINGS: The incidence of appendiceal endometriosis is highly variable depending on the patient population selected. Although rare in patients undergoing appendectomy for acute appendicitis, women with endometriosis may experience rates as high as 9.3-39.0%, especially when suffering from deep infiltrative endometriosis. Appendectomy may also significantly reduce pain in women with unexplained chronic pelvic pain. SUMMARY: Despite lack of prospective data, retrospective studies suggest that appendectomy during gynecologic procedures for chronic pelvic pain and severe endometriosis may be beneficial and necessary to fully address the treatment of these complex gynecologic conditions. In these clinical scenarios, the benefits of laparoscopic appendectomy at the time of the primary gynecologic procedure may outweigh the risks and cost, and should be discussed with patients preoperatively.

Uterine-sparing Laparoscopic Resection of Accessory Cavitated Uterine Masses.

STUDY OBJECTIVE: To demonstrate surgical techniques utilized during uterine-sparing laparoscopic resections of accessory cavitated uterine masses (ACUMs). ACUMs represent a rare uterine entity observed in premenopausal women suffering from dysmenorrhea and recurrent pelvic pain. The diagnosis is made when an isolated extra-cavitated uterine mass is resected from an otherwise normal appearing uterus with unremarkable endometrial lumen and adnexal structures. Pathologic confirmation requires an accessory cavity lined with endometrial epithelium (and corresponding glands and stroma) filled with chocolate-brown fluid. Adenomyosis must be absent. Although the origin of ACUMs is currently unknown, the most common presentation is a 2-4 cm lateral uterine wall mass at the level of the insertion of the round ligament. Hence it has been hypothesized that gubernaculum dysfunction may be responsible for duplication or persistence of paramesonephric tissue leading to ACUM formation as a new Müllerian anomaly. DESIGN: A stepwise surgical tutorial describing 2 laparoscopic ACUM resections using a narrated video (Canadian Task Force classification III). SETTING: An academic tertiary care hospital. PATIENTS: In this video, we present 2 patients who underwent uterine-sparing laparoscopic resections of their ACUM in order to preserve fertility (Case 1) or avoid the complications and surgical recovery time of a total laparoscopic hysterectomy (Case 2). Case 1 is a 19-year-old, gravida 0, para 0 woman with dysmenorrhea and recurrent pelvic pain who presented for multiple emergency room and outpatient evaluations. Transvaginal ultrasonography was unremarkable except for a 28×30×26mm left lateral uterine mass with peripheral vascular flow that was initially felt to be a leiomyoma or rudimentary uterine horn. MRI imaging, however, demonstrated this mass to be more consistent with an ACUM. This was based on the lack of communication between the lesion and the main uterine cavity exhibited by high T2 signal (compatible with endometrial tissue) surrounding low T2/high T1 signal in the dependent aspects (representing blood products). After counseling regarding treatment options including medical management with hormonal contraception, the patient elected for definitive fertility preserving laparoscopic resection. In contrast, case 2 is a 39-year-old, gravida 3, para 3 woman with a 2 month history or left lower quadrant pain following her last vaginal delivery. Transvaginal ultrasonography showed a 23×18×19mm cystic structure within the left uterine wall, which was confirmed to represent an ACUM on MRI. Although she had no desire for fertility preservation, the patient elected for surgical resection of the mass as opposed to a hysterectomy in order to minimize complications and recovery time. INTERVENTIONS: Laparoscopic resection of ACUMs in patients desiring uterine preservation. MEASUREMENTS AND MAIN RESULTS: Laparoscopic resection of the ACUMs was performed utilizing 2 different techniques. In both cases, dilute vasopressin was injected with a modified butterfly or spinal needle along the uterine-ACUM serosal interphase to aid with hemostasis. In patients desiring to preserve fertility (case 1) monopolar energy is utilized to make an incision along the ACUM serosa to help facilitate dissection. ACUM enucleation is then commenced in a circumferential manner along the ACUM and uterine myometrial interphase utilizing bipolar energy. In contrast to leiomyomas where dissection advances along the pseudocapsule, ACUM have poorly delineated borders with disorganized muscular fibers making dissection particularly difficult. A variety of instruments can be utilized to help in the sequential circumferential dissection in addition to a bipolar device including a single-tooth tenaculum, myoma hook, suction device or fine-needle grasper. Ultimately, the ACUM is transected off its uterine-myometrial attachment and hemostasis is obtain before closing the uterine defect in at least 2 layers using a 2-0 barbed V-Loc (Medtronic, Minneapolis, MN). If fertility preservation is no longer desired, the dissection can greatly be expedited by performing a salpingectomy and skeletonizing the ACUM from the leaves of the broad ligament (case 2). A monopolar L-hook can then be used to transect the ACUM from the remaining uterine body. While difficult, these cases can be completed laparoscopically in approximately 2 hours with minimal blood loss. CONCLUSIONS: ACUMs are hypothesized to represent a previously under recognized Müllerian anomaly linked to gubernaculum dysfunction that occurs in premenopausal women with dysmenorrhea and chronic pelvic pain. Uterine and fertility sparing laparoscopic resection is possible but challenging due to poorly defined planes.

Characterization and Preoperative Risk Analysis of Leiomyosarcomas at a High-Volume Tertiary Care Center.

INTRODUCTION: Uterine morcellation in minimally invasive surgery has recently come under scrutiny because of inadvertent dissemination of malignant tissue, including leiomyosarcomas commonly mistaken for fibroids. Identification of preoperative risk factors is crucial to ensure that oncologic care is delivered when suspicion for malignancy is high, while offering minimally invasive hysterectomies to the remaining patients. OBJECTIVES: The aim of this study was to characterize risk factors for uterine leiomyosarcomas by reviewing preoperative, intraoperative, and postoperative data with an emphasis on the presence of concurrent fibroids. METHODS: A retrospective case-control study of women undergoing hysterectomy with pathologic diagnosis of uterine leiomyosarcoma at a tertiary care center between January 2005 and April 2014. RESULTS: Thirty-one women were identified with leiomyosarcoma and matched to 124 controls. Cases with leiomyosarcoma were more likely to have undergone menopause and to present with larger uteri (19- vs 9-week sized), with the most common presenting complaint being a pelvic mass (35.5% vs 8.9%). Controls were ten times more likely to have undergone a tubal ligation (30.6% vs 3.2%). Endometrial sampling detected malignancy preoperatively in only 50% of cases. Leiomyosarcomas were more commonly present when pelvic masses were identified in addition to fibroids on preoperative imaging. Most leiomyosarcoma cases (77.4%) were performed by oncologists via an abdominal approach (83.9%), with only 2 of 31 leiomyosarcomas being morcellated. Comparative analysis of preoperative imaging and postoperative pathology showed that in patients with leiomyosarcoma, fibroids were misdiagnosed 58.1% of the time, and leiomyosarcomas arose directly from fibroids in only 6.5% of cases. CONCLUSIONS: Leiomyosarcoma risk factors include older age/postmenopausal status, enlarged uteri of greater than 10 weeks, and lack of previous tubal ligation. Preoperative testing failed to definitively identify leiomyosarcomas, although the presence of synchronous pelvic masses in fibroid uteri should raise clinical suspicion. Given the difficulty of preoperative identification, future efforts should focus on the development of safer minimally invasive techniques for uterine morcellation.

Hemostasis During Ovarian Cystectomy: Systematic Review of the Impact of Suturing Versus Surgical Energy on Ovarian Function.

This systematic review compares the effect of suturing and surgical energy used for hemostasis during ovarian cystectomies on ovarian function. A search of Scopus, Embase, and PubMed databases was conducted through December 1, 2016 for prospective, retrospective, and randomized controlled trials that analyzed ovarian function after ovarian cystectomies where hemostasis was obtained using suturing versus surgical energy. Of the 25 studies identified, 12 with a total of 1133 subjects met the criteria and were included in this review. Analysis of the pooled data strongly supports the use of suturing rather than surgical energy (bipolar or ultrasonic coagulation) for hemostasis, because it provides improved preservation of ovarian function at the time of cystectomy. Four of 8 ovarian reserve markers (anti-Müllerian hormone, antral follicle count, peak systolic velocity, and ovarian volume) demonstrated a positive association using suturing, whereas the remainder of ovarian markers showed a positive trend toward suturing or noninferiority to bipolar energy. In conclusion, suturing for hemostasis after ovarian cystectomy is superior to surgical energy in preserving ovarian function. Further studies are needed to assess whether this difference is clinically relevant in regards to fertility and premature ovarian failure. (USPSTF Level II-1 Evidence).

Anatomic vascular considerations in uterine artery ligation at its origin during laparoscopic hysterectomies.

Pelvic pathology such as fibroids, endometriosis, adhesions from previous pelvic surgeries, or ovarian remnants can distort anatomy and pose technical challenges during laparoscopic hysterectomies. Retroperitoneal dissection to ligate the uterine artery at its vascular origin can circumvent these obstacles, resulting in a safer procedure. However, detailed anatomic knowledge of the course of the uterine artery and understanding of vascular variations are essential for optimal dissection. We frequently encounter a C-shaped uterine artery variation during retroperitoneal dissection. We describe the key steps in identification and isolation of this variant, approaching the uterine artery origin either from the pararectal space or by utilizing the medial umbilical ligament coursing through the paravesical space. We also review other known uterine artery configurations. These techniques allow for safe completion of complex laparoscopic hysterectomies performed for various gynecologic diseases.

Impact of esophagogastroduodenoscopy and ileocolonoscopy on diagnosis and therapy in patients with rheumatic diseases-a retrospective cohort study.

OBJECTIVES: Many rheumatic diseases as well as their medications may cause gastrointestinal (GI) pathologies; in addition, some primary GI diseases may contribute or lead to rheumatic disease manifestations. The aim of this study is to analyze the clinical relevance of esophagogastroduodenoscopy (EGD) and ileocolonoscopy (IC) in patients suffering from inflammatory rheumatic diseases. METHODS: A retrospective chart review was performed for all rheumatological inpatients who underwent EGD and/or IC within 2 years. RESULTS: Within 2 years, 456 patients (261 female, 195 male) underwent 752 endoscopic investigations of the GI tract (419 EGDs and 333 ICs). Of all patients, 152 (33.3%) did not report any GI complaints. However, 28 of these asymptomatic patients (18.4%) suffered from esophagitis, a gastric ulcer could be identified in 20 patients (13%), whereas unspecific colitis was diagnosed in 19 patients (12.5%). In addition, 14 patients (9.2%) suffered from clinically unapparent Crohn's disease and two patients from Whipple's disease. In one patient with polymyalgia rheumatica, colon cancer was diagnosed. Altogether 304 patients reported GI complaints. Of these, 292 (39%) endoscopic investigations had impact on the final diagnosis or therapeutic strategy. The antirheumatic medication or the concomitant medication was changed in 18% of the patients due to the endoscopic findings; in 29 patients (6.5%) the initially clinically presumed diagnosis had to be corrected. In 70 patients (15%) with an undefined rheumatic diagnosis prior to endoscopy, endoscopic findings were decisive to establish the final diagnosis. CONCLUSION: EGD and IC have a high diagnostic impact on patients with rheumatic diseases presenting with or without concomitant GI symptoms.

Referral patterns for pelvic floor surgical prosthesis complications: from symptom onset and initial treatments to evaluation at a tertiary care center.

UNLABELLED: Although complications of prosthesis products used for pelvic organ prolapse and stress urinary incontinence have been extensively characterized, little is known about their referral patterns and the time lapse from symptom onset to evaluation and treatment. The aim of our study was to retrospectively describe the patterns and timing of referral of prosthesis-related complications and initial prereferral treatments. METHODS: We retrospectively identified patients referred to the University of Virginia from January 2002 to October 2012 with prosthesis complications related to previously placed devices for pelvic floor disorders. RESULTS: We identified 55 prosthesis-related complications in 51 patients. The types of prostheses used included mesh or permanent suture for pelvic organ prolapse (vaginal prolapse, n = 24), tapes for stress urinary incontinence (midurethral, n = 21), and materials for abdominal sacrocolpopexies and vaginal vault suspensions (intraperitoneal, n = 10). Original surgeons, who most frequently were general obstetric/gynecology providers, were the referring physicians for only 3.6% of the time. Vaginal and midurethral prosthesis complications occurred most commonly in the early postoperative period, whereas intraperitoneal were more commonly delayed. Patients reported to have experienced adverse symptoms on average from several months up to years while receiving conservative therapies by secondary providers and/or surgical treatments with their original surgeon before referral to our institution. CONCLUSIONS: Referral of prosthesis-related complications is often delayed, and most cases required evaluation by a secondary provider other than the original surgeon before a referral was initiated. Timing of symptom onset was dependent on the prosthesis type with up to 1 in 3 having a late presentation that may in part be delayed by prereferral treatments. Further education of original surgeons, primary care providers, and patients on the presentation, timing, and outcomes of potential prosthesis-related complications is needed to facilitate earlier management at tertiary care centers.

Induced pluripotent stem cells from familial Alzheimer's disease patients differentiate into mature neurons with amyloidogenic properties.

Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in ß-amyloid (Aß)42/Aß40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.

Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.

Short-term surgical outcomes and characteristics of patients with mesh complications from pelvic organ prolapse and stress urinary incontinence surgery.

INTRODUCTION AND HYPOTHESIS: Surgical treatment of pelvic organ prolapse (POP) and stress urinary incontinence (SUI) can include the use of synthetic materials. Placement of synthetic materials into the vaginal wall, through either the vagina or the abdomen, includes the risk of complications such as vaginal wall extrusion or pain. There is little data regarding outcomes following treatment of mesh complications. METHODS: A retrospective chart review of patients who underwent excision of mesh placed for POP or SUI between 1 January 2001 and 31 October 2012 was performed at the University of Virginia. Chart abstraction queried patient demographics, clinical history, physical examination, pre- and post-excision symptoms, and operative findings. The International Continence Society (ICS) and International Urogynecological Association (IUGA) classification system was used to define the nature and location of mesh complications. RESULTS: A total of 57 patients (26 mid-urethral slings, 23 transvaginal prolapse, 9 intraperitoneal prolapse) with the diagnosis of mesh extrusion into the vaginal wall were analyzed. Twenty-five (average 2.8 cases/year) original mesh surgeries occurred between January 2001 and January 2010 and 41 (average 20.5 cases/year) occurred after January 2010. The most common presenting patient complaints were chronic pelvic pain (55.9 %), dyspareunia (54.4 %), and vaginal discharge (30.9 %). At a 6-week post-operative visit, 57.3 % of patient's symptoms were completely resolved and 14.6 % were improved. CONCLUSION: Clinicians should be cognizant of the variable presentations of post-operative vaginal mesh complications. Mesh excision by experienced pelvic surgeons is an effective and safe treatment for these complications; however, a significant number of patients may have persistent symptoms following surgery.

Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (~0.001%-0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ~50% in purified episome-expressing cells. Lineage-committed CD33(+)CD45(+)CD34(-) myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG(+)TRA-1-81(+) hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC.

A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.

BACKGROUND: The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods. METHODOLOGY/PRINCIPAL FINDINGS: We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. CONCLUSION/SIGNIFICANCE: This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation.

Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.

Challenges and strategies for generating therapeutic patient-specific hemangioblasts and hematopoietic stem cells from human pluripotent stem cells.

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.