Background Chromosomal microarray (CMA) has been shown to be cost-effective over karyotyping in invasive prenatal diagnosis for pregnancies with fetal ultrasound anomalies. Yet, information regarding preceding and subsequent tests must be considered as a whole before the true cost-effectiveness can emerge. Currently in Hong Kong, karyotyping is offered free as the standard prenatal test while genome-wide array comparative genome hybridization (aCGH), a form of CMA, is self-financed. A new algorithm was proposed to use aCGH following quantitative fluorescent polymerase chain reaction (QF-PCR) as primary test instead of karyotyping. This study aims to evaluate the cost-effectiveness of the proposed algorithm versus the current algorithm for prenatal diagnosis in Hong Kong. Methods Between November 2014 and February 2016, 129 pregnant women who required invasive prenatal diagnosis at two public hospitals in Hong Kong were prospectively recruited. The proposed algorithm was performed for all participants in this demonstration study. For the cost-effectiveness analysis, cost and outcome (diagnostic rate) data were compared with that of a hypothetical scenario representing the current algorithm. Further analysis was performed to incorporate women’s willingness-to-pay for the aCGH test. Impact of government subsidies on the aCGH test was explored as a sensitivity analysis. Results The proposed algorithm dominated the current algorithm for prenatal diagnosis. Both algorithms were equally effective but the proposed algorithm was significantly cheaper ( p ≤ 0.05). Taking into account women’s willingness-to-pay for an aCGH test, the proposed algorithm was more effective and less costly than the current algorithm. When the government subsidy reaches 100%, the maximum number of diagnoses could be made. Conclusion By switching to the proposed algorithm, cost saving can be achieved whilst maximizing the diagnostic rate for invasive prenatal diagnosis. It is recommended to implement aCGH as a primary test following QF-PCR to replace the majority of karyotyping for prenatal diagnosis in Hong Kong.

Chromosomal microarray (CMA) is recommended as a first tier investigation for patients with developmental delay (DD), intellectual disability (ID), autistic spectrum disorder (ASD), and multiple congenital anomalies (MCA). It is widely used in the prenatal and postnatal settings for detection of chromosomal aberrations. This is a retrospective review of all array comparative genomic hybridization (aCGH/ array CGH) findings ascertained in two major prenatal and postnatal genetic diagnostic centers in Hong Kong from June 2012 to December 2017. Medical records were reviewed for cases with pathogenic and variants of uncertain clinical significance (VUS). Classification of copy number variants (CNVs) was based on current knowledge and experience by August 2018. The aims of this review are to study the diagnostic yield of array CGH application in prenatal and postnatal settings in Hong Kong and to describe the spectrum of abnormalities found. Prenatal indications included abnormal ultrasound findings, positive Down syndrome screening, abnormal noninvasive prenatal test results, advanced maternal age and family history of chromosomal or genetic abnormalities. Postnatal indications included unexplained DD, ID, ASD, and MCA. A total of 1,261 prenatal subjects and 3,096 postnatal patients were reviewed. The prenatal diagnostic yield of pathogenic CNV and VUS (excluding those detectable by karyotype) was 3.5%. The postnatal diagnostic yield of pathogenic CNV was 15.2%. The detection rates for well-defined microdeletion and microduplication syndromes were 4.6% in prenatal and 6.1% (1 in 16 index patients) in postnatal cases, respectively. Chromosomes 15, 16, and 22 accounted for over 21 and 25% of pathogenic CNVs detected in prenatal and postnatal cohorts, respectively. This review provides the first large scale overview of genomic imbalance of mostly Chinese patients in prenatal and postnatal settings.

A fetus of Chinese descent presented with ultrasound features of anemia at 20 weeks' gestation. Father had low a mean corpuscular volume (MCV) level. Multiplex gap-polymerase chain reaction (gap-PCR) excluded common -thalassemia (-thal) deletions and mutations and PCR sequencing of the 1- and 2-globin genes were negative. The fetus had a normal karyotype. Array comparative genomic hybridization (aCGH) showed a single copy loss of 189.87kb in chromosome 11p15.4, involving the whole -globin gene cluster, inherited from the father. Multiplex ligation-dependent probe amplification (MLPA) confirmed the deletion included the epsilon-globin gene, confirming the diagnosis of heterozygous (epsilon)(0)-thalassemia [(epsilon)(0)-thal], also inherited from the father. The fetus had a worsening anemic condition in utero and required a transfusion at 26 weeks' gestation, raising the hemoglobin (Hb) level from 5.3 to 12.6g/dL. A cesarean-section was subsequently performed at 32 weeks' gestation because of reduced fetal movements, and a 1650g baby girl with good Apgar scores was delivered. Hemoglobin at birth was 12.8g/dL, gradually dropping to 6.8g/dL, requiring three neonatal transfusions. Her condition gradually stabilized after 2 months with Hb stable at 8.0g/dL. Family screening by MLPA showed that the paternal grandmother carried the same deletion. The deletion in this case is distinct and is the reported first case. The deletion transmitted across three successive generations with great phenotypic variation. The final adult phenotype of (epsilon)(0)-thal is usually mild, therefore, with accurate prenatal diagnosis this condition is salvageable by in utero and early neonatal transfusions, preventing adverse pregnancy and neonatal outcomes.

Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes.

Hydrops fetalis is commonly due to Hb Bart's (gamma(4)) disease in South East Asia. Here, we report an unusual case of hydrops fetalis due to congenital dyserythropoietic anemia (CDA) associated with compound heterozygosity for Kruppel-like factor 1 (KLF1) gene mutations. Fetal cardiomegaly was first detected on routine mid- trimester scan in a pregnant woman with normal mean corpuscular volume (MCV) and Rhesus positive status. The fetus subsequently developed hydrops fetalis, and cordocentesis showed severe fetal anemia with a hemoglobin (Hb) level of 3.4 g/dL. Common causes of fetal anemia including Hb Bart's disease, parvovirus infection, and red cell antibodies were excluded. In view of the marked increase in erythroblasts at various stages of erythropoiesis, the diagnosis of CDA was suspected. We screened the couple for previously reported KLF1 gene mutations, showing that the mother was heterozygous for the c. 525_526insCGGCGCC, p. Gly176Argfs * 179 mutation, and her husband heterozygous for c. 1012C> A, p.Pro338Thr mutation. The fetus was a compound heterozygote for these two KLF1 mutations. After counseling, repeated intrauterine transfusions were given at 27, 29, and 34 weeks' gestation; the hydrops fetalis was resolved. The baby was delivered at 34 weeks' gestation and required monthly blood transfusions but was otherwise thriving. Bone marrow aspiration at 10 months of age showed the features of ineffective erythropoiesis, compatible with CDA. In conclusion, hydrops fetalis can rarely be due to CDA associated with a compound heterozygous mutation for KLF1 gene mutations, and be managed by repeated intrauterine transfusions. Our present report adds to the wide clinical spectrum of KLF1 mutations.

Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers—CD34, neuropilin 1, and human kinase insert domain-containing receptor—and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

The field of regenerative medicine encompasses a breathtaking array of interdisciplinary scientific approaches with the promise of delivering future therapies to meet current unmet medical needs for patients. Increasingly more of these innovative products are being translated into human clinical trials in the United States, and general familiarity of the FDA is important to efficiently navigate the process. The basics of FDA history, organization, and processes are described herein for those new to clinical translation, with more detailed content added regarding approval pathways, regulations, guidances, and select special topics of relevance to regenerative medicine. In addition to the cumulative experience of previous products, the FDA regulatory approach to medical products evaluation includes an ongoing assessment of how the science of those products informs regulatory policy. FDA engages in ongoing dialogue with the scientific community and product sponsors to continue to develop science-based regulatory review policies that are robust and predictable in order to meet the needs of the challenging array of products that are on the horizon.

We here report an unusual case of Hb Bart's (γ4) disease. Thalassemia screening of a couple showed that the wife was an α(0)-thalassemia (α(0)-thal) carrier and her husband's mean corpuscular volume (MCV) was normal. Chorionic villus sampling (CVS) was performed at 13 weeks' gestation for positive Down syndrome screening and chromosomal study of the cultured CVS showed a normal karyotype. Ultrasound examination at 22 weeks' gestation showed fetal cardiomegaly and raised middle cerebral artery peak systolic velocity. Cordocentesis confirmed fetal anemia and showed Hb Bart's disease. Multiplex gap-polymerase chain reaction (gap-PCR) for α-thal deletions on DNA extracted from the CVS showed the presence of a homozygous α(0)-thal - -(SEA) (Southeast Asian) deletion. The husband was found to be a carrier of the α(+)-thal -α(3.7) (rightward) deletion. Non paternity was excluded by fluorescent PCR using short tandem repeat (STR) markers on chromosomes 13, 18 and 21. A de novo terminal deletion of chromosome 16 was excluded by array comparative genomic hybridization (aCGH). Detection of uniparental disomy (UPD), using STR markers on chromosome 16 showed maternal uniparental isodisomy from 16pter to 16p13.2, and uniparental heterodisomy from 16p13.13 to 16qter.

FDA recently released a draft-guidance on the preclinical assessment of investigational cellular and gene therapy products.

The U.S. Food and Drug Administration applies regulatory flexibility to balance benefits and risks to subjects in cell-therapy clinical trials.

U.S. Food and Drug Administration applies regulatory flexibility to balance benefits and risks to subjects in cell-therapy clinical trials.

Rapid blood perfusion is critical for postimplantation survival of thick, prevascularized bioartificial tissues. Yet the mechanism by which implanted vascular networks inosculate, or anastomose, with the host vasculature has been unknown, making it difficult to develop optimized strategies for facilitating perfusion. Here we show that implanted vascular networks anastomose with host vessels through a previously unidentified process of "wrapping and tapping" between the engrafted endothelial cells (ECs) and the host vasculature. At the host-implant interface, implanted ECs first wrap around nearby host vessels and then cause basement membrane and pericyte reorganization and localized displacement of the underlying host endothelium. In this way, the implanted ECs replace segments of host vessels to divert blood flow to the developing implanted vascular network. The process is facilitated by high levels of matrix metalloproteinase-14 and matrix metalloproteinase-9 expressed by the wrapping ECs. These findings open the door to new strategies for improving perfusion of tissue grafts and may have implications for other physiologic and pathologic processes involving postnatal vasculogenesis.

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A major obstacle in tissue engineering is poor post‐implantation cell survival due to the lack of blood perfusion. One approach is to populate the graft before implantation with endothelial or progenitor cells that can quickly organize into a network and connect with the host vasculature to achieve blood perfusion. Many types of endothelial and endothelial progenitor cells are capable of this, but the mechanism by which the implanted vascular networks anastomose with the host vasculature is unknown. Here we show that it occurs through a previously unreported process of “wrapping and tapping” (WAT). At the host‐implant interface, implanted ECs wrap around nearby host vessels, cause host vessel regression by disrupting their pericytes and basement membrane, and then completely replace segments of host vessels to divert flow into the contiguous, engrafted vascular network. This process is facilitated by high levels of MMP‐14 and MMP‐9 expressed specifically by wrapping ECs. Our results provide mechanistic understanding on this critical step of vascularization and may have implications for other physiological and pathological processes involving post‐natal vasculogenesis.

Therapeutic revascularization with either exogenous angiogenic growth factors or vascular cells has yet to demonstrate efficacy in the clinic. Injection of angiogenic growth factors often produces unstable and abnormal blood vessels. Blood vascular networks derived from implanted endothelial cells persist only transiently due to the insufficient recruitment of perivascular cells. We hypothesize that a combination of the two approaches may act synergistically to yield a better result. To enhance the recruitment of perivascular cells, human umbilical vein endothelial cells were genetically modified to overexpress platelet-derived growth factor (PDGF)-BB. PDGF-BB overexpression promoted both proliferation and migration of perivascular precursor cells (10T1/2 cells) in vitro. When mock-infected endothelial cells were implanted alone in vivo, they formed transient blood vascular networks that regressed by day 30. PDGF-BB overexpression enhanced the survival of endothelial cells in vivo. However, the PDGF-BB-expressing vessel network failed to establish patent blood flow. Co-implantation of PDGF-BB-overexpressing endothelial cells with 10T1/2 cells paradoxically resulted in the rapid regression of the vascular networks in vivo. PDGF-BB stimulated the expression of both chemokine (C-C motif) ligand 2 (CCL2) and CCL7 in 10T1/2 cells and led to the increased accumulation of macrophages in vivo. These results suggest a potential negative interaction between angiogenic growth factors and vascular cells; their use in combination should be carefully tested in vivo for such opposing effects.

Background Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses. Methodology/Principal Findings We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R2 = 0.84) and urine (R2 = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2. Conclusion/Significance We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.

Tissue engineering requires formation of a de novo stable vascular network. Because of their ability to proliferate, differentiate into endothelial cells, and form new vessels, blood-derived endothelial progenitor cells (EPCs) are attractive source of cells for use in engineering blood vessels. However, the durability and function of EPC-derived vessels implanted in vivo are unclear. To this end, we directly compared formation and functions of tissue-engineered blood vessels generated by peripheral blood- and umbilical cord blood-derived EPCs in a model of in vivo vasculogenesis. We found that adult peripheral blood EPCs form blood vessels that are unstable and regress within 3 weeks. In contrast, umbilical cord blood EPCs form normal-functioning blood vessels that last for more than 4 months. These vessels exhibit normal blood flow, perm-selectivity to macromolecules, and induction of leukocyte-endothelial interactions in response to cytokine activation similar to normal vessels. Thus, umbilical cord blood EPCs hold great therapeutic potential, and their use should be pursued for vascular engineering.