Feeder-free System Research Articles

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, during which unlimited spermatozoa is produced daily derived from SSCs in the testis throughout life of the male. Germline stem (GS) cells can be isolated from spermatogonia, which shared the characteristics of SSCs and embryonic stem cells (ESCs), and can be passaged stably in vitro. The study of GS cells contributes to understanding spermatogenesis process. However, little is known about the GS cells in domestic animals. Here, we report the successful establishment of a serum- and feeder-free system for multipotent male GS cells (mGSCs) from postnatal porcine testis. These cells expressed pluripotent markers, such as Oct4, Nanog, C-myc, and germline-specific markers including Vasa, CD90, CD49f, Gfrα1, Plzf and Dazl. Then we assayed the developmental potential of these cells in vitro. The porcine multipotent male germline stem cells (pmGSCs) can form embryoid bodies (EBs) by suspension culture. Immunofluorescence analysis showed that the EBs differentiated into neuron-specific enolase (NSE, ectoderm), α-actin (mesoderm), and Pdx1 (endoderm) positive cells. These cells induced by 10-6 M retinoic acid (RA) could be differentiated into spermatid-like cells which were positive for Acrosin. The pmGSCs has been cultured over 14 passages. Thus, we have established a long-term culture system for pmGSCs. This culture system provides a platform for the study of porcine mGSCs.

SIRT2 Plays Essential Role in Early Hematopoiesis through Deacetylation of LMO2 Protein

Nicotinamide phosphoribosyltransferase (NAMPT) is known as a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) and supplies NAD+ to sirtuins (SIRTs) that require NAD+ to activate their protein deacetylase functions. SIRTs are members of the NAD+-dependent class III histone deacetylase family. We already reported that NAMPT-NAD+-SIRT1 pathway is essential for granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis (Skokowa J, et al. Nat Med. 2009).In the present study, we analyzed the mechanism of NAMPT-NAD+ -SIRTs-triggered hematopoiesis and myelopoiesis using human induced pluripotent stem (iPS) cells model. We applied a serum- and feeder-free monolayer hematopoietic differentiation system of iPS cells by sequential incubation of iPS cells with combinations of mesoderm- and hematopoiesis-inducing cytokines. We found that addition of NAMPT to granulopoiesis-inducing iPS cells culture medium resulted in a marked increase of CD15+ and CD16+ myeloid and granulocytic cell numbers. These results are compatible with our published data in healthy donor derived CD34+ cells. To evaluate at which stage of hematopoietic differentiation NAMPT plays a role, we cultured iPS cells in the presence or absence of the specific inhibitor of NAMPT, FK866. Interestingly, we found marked reduction of VEGFR2+ CD34+ early hematopoietic progenitor cells at day 6 of differentiation after inhibition of NAMPT. These data argue for the requirement of NAMPT and its downstream effectors at early stages of hematopoietic differentiation. To evaluate which of the SIRTs is essential in this process, we tested early hematopoietic differentiation of iPS cells in the presence of specific inhibitors of several SIRTs. We found that inhibition of SIRT2 using its specific inhibitor AC93253 significantly suppressed differentiation into VEGFR2+ CD34+ early hematopoietic progenitor fraction. Furthermore, the emergence of CD43+ hematopoietic progenitor cells at day 13 was completely suppressed after inhibition of SIRT2. These results indicate that NAMPT-NAD+-SIRT2 signaling is needed for early hematopoiesis. We further evaluated downstream targets of NAMPT-NAD+-SIRT2. qRT-PCR analysis of well-known early hematopoietic genes (GATA2, RUNX1, LMO2, TAL1) revealed that mRNA expression of these factors was not down-regulated after NAMPT or SIRT2 inhibition. We therefore tested whether hematopoietic functions of these proteins might be affected by post-transcriptional modifications via NAMPT-NAD+-SIRT2-triggered deacetylation. We tested protein-protein interaction between SIRT2 and these candidate proteins in differentiated iPS cells on day 6 of culture using Duolink technique, which allows identification of protein complexes in single cells. We found that RUNX1, LMO2 and TAL1 proteins interact with SIRT2 protein. To identify if these proteins could be deacetylated by SIRT2, we compared the signals from total- and de-/acetylated- candidate proteins in differentiated iPS cells on day6 treated or not with SIRT2 inhibitor using Duolink-FACS, Duolink technique in combination with flow cytometry, as a readout method. This analysis revealed that only LMO2 is deacetylated by SIRT2. LMO2 is known as an essential transcriptional regulator in early hematopoiesis. It is a component of TAL1 complex, which consist of LMO2, TAL1, LDB1, E47 and GATA1/2 proteins and has transcriptional activity as a complex on hematopoiesis-specific target genes as KIT, GATA1, GYPA, KLF1 and KDR. Interestingly, mRNA expression levels of all these genes were markedly downregulated after inhibition of NAMPT or SIRT2, which was in line with severely reduced numbers of early hematopoietic progenitors.Overall, these results indicate that NAMPT, known to be essential for myeloid differentiation, induces early stages of hematopoietic differentiation by SIRT2-triggered deacetylation of LMO2. DisclosuresNo relevant conflicts of interest to declare.

Cooperative Effect of Erythropoietin and TGF-β Inhibition on Erythroid Development in Human Pluripotent Stem Cells.

Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34(+)CD31(+) cells derived from human pluripotent stem cells (hPSCs) contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34(+)CD31(+) progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34(+)CD31(+) progenitor cells into CD235a(+) erythroid cells through CD41(+)CD235a(+) megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34(+)CD31(+) progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-β) inhibits the development of CD34(+)CD31(+) progenitor cells. Apoptosis of progenitor cells is induced by TGF-β in early erythroid differentiation. Suppression of TGF-β signaling by SB431542 at early stage of CD34(+)CD31(+) progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-β suppression and EPO stimulation promote erythropoiesis of CD34(+)CD31(+) progenitor cells derived from hPSCs.

Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×105 seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34+ cells, we obtained 148 iPS colonies from 105 seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.

Feeder-free and xeno-free culture of human pluripotent stem cells using UCBS matrix.

The ideal medium for human pluripotent stem cells (hPSCs) culture should be feeder-free, xeno-free, and completely defined. The present study aims to establish a new feeder-free and xeno-free system for culturing hPSCs. The system consists of the matrix, which was prepared from human umbilical cord blood serum (UCBS) and used to coat the culture plates, and the xeno-free medium, which was conventional serum-free hES medium supplemented with high concentrations of bFGF and Fibronectin. Compared with matrigel and mouse embryonic fibroblasts (MEFs), the UCBS matrix was proved to be equally suitable for the growth of hPSCs. After a series of experiments with different media and cytokins, the UCBS matrix was found worked the best with the basic medium (BM) supplemented with 20 ng/mL bFGF, 10 ug/mL fibronectin and Y-27632 for culture of hES cells. The hPSCs maintained normal karyotype, high proliferation rate and self-renewal ability after continuous culture more than 10 passages in this feeder-free and xeno-free system. Furthermore, a new human embryonic stem (hES) cell line was derived from discarded day 3 embryos in this newly developed culture system. In conclusion, this feeder-free and xeno-free system could not only be used to the culture hPSCs, but could also be used to derive new hES cell lines.

Direct Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells by OCT4.

: Cellular reprogramming or conversion is a promising strategy to generate desired stem cell types from somatic cells. Neural stem cells (NSCs) have the potential to regenerate central nervous system tissue and repair damage in response to injury. However, NSCs are difficult to isolate from human tissues and expand in sufficient quantities for therapy. Here, we report a method to generate neural stem cells from cord blood CD34-positive cells by ectopic expression of OCT4 in a feeder-free system. The induced cells (iNSCs) show a characteristic NSC-like morphology and can be expanded in vitro for more than 20 passages. In addition, the iNSCs are positive for neural stem cell-specific markers such as Nestin and Musashi-1 and are similar in gene expression patterns to a human neural stem cell line. The iNSCs express distinct transcriptional factors for forebrain, hindbrain, and spinal cord regions. Upon differentiation, the iNSCs are able to commit into multilineage mature neural cells. Following in vivo introduction into NOD/SCID mice, iNSCs can survive and differentiate in the mouse brain 3 months post-transplantation. Alternatively, we were also able to derive iNSCs with an episomal vector expressing OCT4. Our results suggest a novel, efficient approach to generate neural precursor cells that can be potentially used in drug discovery or regenerative medicine for neurological disease and injury. This study describes a novel method to generate expandable induced neural stem cells from human cord blood cells in a feeder-free system by a single factor, OCT4. The data are promising for future applications that require the generation of large amounts of autologous neural stem cells in disease modeling and regenerative medicine.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Effects of Feeder Layers, Culture Media, Conditional Media, Growth Factors, and Passages Number on Stem Cell Optimization

Stem cells are undifferentiated and self-renewal cells which could be obtained from the body or artificially derived from an adult somatic cell by forced expression of specific genes. In recent years stem cells are widely used in laboratory for tissue engineering and therapeutic applications. There are different factors and conditions that affect the stem cell culture such as feeder layers, atmosphere, kind of medium, growth factors, passages number, and conditional media with animal or human sources. Optimization of stem cell culture for medical approaches and regenerative medicine is important. Therefore, in the present study, the effect of these factors and agents on optimization of stem cell culture has been discussed. This review study showed that optimization of feeder layer, atmosphere, and using supplemented media with essential growth factors could help in maintaining the stem cells in undifferentiated state in vitro. The present study indicated that optimization of stem cell culture depends on the kind of each cell type and using stem cells in low passage number could decrease chromosomal abnormalities and DNA damages. For inhibiting the stem cell contamination by feeder cell lines, culture of these cells on feeder free systems like Matrigel matrix in conditioned media supplemented with essential growth factors is useful. Also, for eradicating immune system responses and reducing the risk of animal pathogen transfer, culture of stem cells on human feeder in optimized media is suitable for therapeutic approaches and regenerative medicine.

The effect of isolation and culture methods on epithelial stem cell populations and their progeny—toward an improved cell expansion protocol for clinical application

Background aimsThe use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. MethodsWe compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. ResultsDuring the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. Discussion.Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.

Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.

Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system.

ObjectivesKidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system.Materials and MethodsWe designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility.ResultsAfter modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems.ConclusionsOur novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.

Induced Pluripotent Stem Cells from Human Placental Chorion for Perinatal Tissue Engineering Applications

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly, we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4, SOX2, KLF4, and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology, immunocytochemistry, and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives, including neural stem cells, beating cardiomyocytes, and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

FDA-Approved Pharmacological Agents, Romiplostium and Oprelvekin, Synergistically Promote Megakaryocytic Differentiation From Human iPSCs In a Chemically Defined System

Platelets, anucleate cells derived from megakaryocytes (MKs) that are generated within the bone marrow, play an important role in the process of physiological hemostasis and in vascular repair. Low platelets in the blood stream result in bleeding risk in thrombocytopenic patients with liver failure, leukemia, or undergoing chemotherapy. Platelet transfusions remain the mainstay of treatment and require a constant supply of platelets. Because platelets from donor blood have a short life-span (only few days in storage), platelets are always in a short supply. In vitro generation of MKs and platelets from human induced pluripotent stem cells (hiPSCs) would provide a patient-specific renewable cell source of MKs and platelets to treat thrombocytopenic patients at risk of hemorrhage. We derived integration-free hiPSCs from peripheral blood cells of more than 20 individuals, and examined two methods of in vitro differentiation into MKs: i) co-culture on 10T1/2 cells or OP9 cells first developed by Takayama et al. (2010), and ii) a feeder-free and serum-free system by first forming embryoid bodies (EBs) in a chemically defined condition, similar to the recently published method of Pick et al. (2013). Although both methods gave rise with similar efficiency to CD41a+CD42a+ MKs with large cell size and high-ploidy DNA, we chose to focus on the feeder-free system that began with EB formation with centrifugal aggregation of hiPSCs (spin-EBs) because it is cheaper, faster, easier to scale up, and represents a chemically defined system. To investigate the effect of growth factors on hiPSC differentiation to MKs, we modified the spin-EB system to three steps: i) mesoderm induction and hematopoietic commitment in the presence of BMP4, VEGF, bFGF and SCF (day 0 to day 11), ii) hematopoietic progenitor and MK differentiation by adding TPO (day 11 to 14), and iii) MK maturation (day 14 to 19). To assess whether the FDA-approved pharmacological agent, Romiplostium (Nplate®, TPO analog), has a similar effect to TPO on MK differentiation from hiPSCs, we isolated hematopoietic progenitor cells at day 14, and differentiated them into MKs with Romiplostium or TPO. Our data demonstrated that Romiplostium (50 ng/ml) gave a 3-fold increase of CD41a+CD42a+ MKs, with similar dose-dependent kinetics as TPO. IL-11 has also been reported to enhance MK development. To test whether FDA-approved pharmacological IL-11, Oprelvekin (Neumega®), further stimulated MK differentiation from hiPSCs, we cultured hematopoietic progenitor cells from day 14 in the presence of Romiplostium and Oprelvekin for 5 days. Our data showed that Romiplostium and Oprelvekin synergistically promote megakaryocytic differentiation. In the presence of Romiplostium, 60 to 95 % of cells were CD41a+CD42a+ MKs. Addition of Oprelvekin significantly increased the number of CD41a+CD42a+ MKs, but not the percentage of CD41a+CD42a+ MKs, suggesting that Oprelvekin enhanced a proliferation of MK progenitors. So far, 10 hiPSC lines from several individuals have been tested using the combination of Romiplostium and Oprelvekin in the feeder-free and serum-free differentiation condition. We are currently investigating if the MKs and platelets generated by this defined and scalable system are as fully functional as those generated from bone marrow CD34+ cells from healthy donors.* The first three authors contributed equally;This study is supported in part by an NIH grant U01 HL-107446 and 2012-MSCRFII-0124 (to ZZ Wang). Disclosures:No relevant conflicts of interest to declare.

The Balance of Positive and Negative Effects of TGF-β Signaling Regulates the Development of Hematopoietic and Endothelial Progenitors in Human Pluripotent Stem Cells

Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34(+)CD31(+)VE-cadherin(+) (VEC(+)) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34(+)CD31(+)VEC(+) cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-β) 1 protein is increased during hPSC differentiation, and that TGF-β signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-β to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34(+)CD31(+)VEC(+) cells. An addition of TGF-β inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34(+)CD31(+)VEC(+) progenitor cells. However, inhibition of TGF-β signaling after mesoderm induction increases CD34(+)CD31(+)VEC(+) progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-β signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

3D microcarrier system for efficient differentiation of human induced pluripotent stem cells into hematopoietic cells without feeders and serum

Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Differentiation of Human Embryonic Stem Cells into Clinically Amenable Keratinocytes in an Autogenic Environment

Human embryonic stem cells (hESCs)-derived keratinocytes hold great clinical and research potential. However, the current techniques are hampered by the use of xenogenic components that limits their clinical application. Here we demonstrated an efficient differentiation of H9 hESCs (H9-hESCs) into keratinocytes (H9-Kert) with the minimum use of animal-derived materials. For differentiation, we established two microenvironment systems originated from H9-hESCs (autogenic microenvironment). These autogenic microenvironment systems consist of an autogenic coculture system (ACC) and an autogenic feeder-free system (AFF). In addition, we showed a stage-specific effect of Activin in promoting keratinocyte differentiation from H9-hESCs while repressing the expression of early neural markers in the ACC system. Furthermore, we also explained the effect of Activin in construction of the AFF system made up of extracellular matrix similar to basement membrane extracted from H9-hESC-derived fibroblasts. H9-Kert differentiated in both systems expressed keratinocyte markers at mRNA and protein levels. H9-Kert were also able to undergo terminal differentiation in high Ca(2+) medium. These findings support the transition toward the establishment of an animal-free microenvironment for successful differentiation of hESCs into keratinocytes for potential clinical application.

Autogenic feeder free system from differentiated mesenchymal progenitor cells, maintains pluripotency of the MEL-1 human embryonic stem cells

Human embryonic stem cells (hESc) are known for its pluripotency and self renewal capability, thus possess great potential in regenerative medicine. However, the lack of suitable xenofree extracellular matrix substrate inhibits further applications or the use of hESc in cell-based therapy. In this study, we described a new differentiation method, which generates a homogeneous population of mesenchymal progenitor cells (hESc–MPC) from hESc via epithelial–mesenchymal transition. The extracellular matrix (ECM) proteins from hESc–MPC had in turn supported the undifferentiated expansion of hESc. Immunocytochemistry and flow cytometry characterization of hESc–MPC revealed the presence of early mesenchymal markers. Tandem mass spectometry analysis of ECM produced by hESc–MPC revealed the presence of a mixture of extracellular proteins which includes tenascin C, fibronectin, and vitronectin. The pluripotency of hESc (MEL-1) cultured on the ECM was maintained as shown by the expression of pluripotent genes (FoxD3, Oct-4, Tdgf1, Sox-2, Nanog, hTERT, Rex1), protein markers (SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, Oct-4) and the ability to differentiate into cells representative of ectoderm, endoderm and mesoderm. In summary, we have established a xeno-free autogenic feeder free system to support undifferentiated expansion of hESc, which could be of clinical relevance.

A novel feeder-free system for human embryonic stem cells and characterization of their sublines with autogenic and allogenic cultivation

We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.