Development of a Xeno-Free Substrate for Human Embryonic Stem Cell Growth

Traditionally, human embryonic stem cells (hESCs) are cultured on inactivated live feeder cells. For clinical application using hESCs, there is a requirement to minimize the risk of contamination with animal components. Extracellular matrix (ECM) derived from feeder cells is the most natural way to provide xeno-free substrates for hESC growth. In this study, we optimized the step-by-step procedure for ECM processing to develop a xeno-free ECM that supports the growth of undifferentiated hESCs. In addition, this newly developed xeno-free substrate can be stored at 4°C and is ready to use upon request, which serves as an easier way to amplify hESCs for clinical applications.

Red Ginseng Extract Facilitates the Early Differentiation of Human Embryonic Stem Cells into Mesendoderm Lineage

Human embryonic stem cells (hESCs) have capacities to self-renew and differentiate into all cell typesin vitro. Red ginseng (RG) is known to have a wide range of pharmacological effectsin vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs), and hESC-derived cardiac progenitors (CPs) were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3βon EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.

Modeling Megakaryopoiesis and Leukemogenesis Using Human and Murine Embryonic Stem Cells

Abstract 2502 Megakaryopoiesis involves the differentiation of progenitor cells into diploid megakaryocytes, which then undergo endomitosis and cytoplasmic maturation resulting in platelet release. Acute Megakaryoblastic Leukemia (AMKL) results when this process goes awry. Nearly one third of pediatric AMKL patients are infants who have the t (1;22) chromosomal translocation (t (1;22)-AMKL) resulting in fusion of the RNA Binding Motif 15 gene (RBM15) on chromosome 1 upstream of the transcriptional cofactor Megakaryoblastic Leukemia 1 gene (MKL1) on chromosome 22. Given that t (1;22)-AMKL primarily affects infants, the leukemia likely originates in utero when the hematopoietic system is in its embryonic stage.Our goal was to establish in vitro methods to study embryonic megakaryopoiesis and leukemogenesis using human and murine embryonic stem cells (hESCs and mESCs). Using the Invitrogen Gateway System, human MKL1 (hMKL1) and human RBM15-MKL1 (hRM) constructs were designed. H9-rtTA, a hESC line constitutively expressing the reverse transcriptional activator (rtTA) under the control of the Ubiquitin C promoter, were transduced with lentivirus harboring either hMKL1 or hRM with an IRES-GFP promoter under the control of the tetracycline responsive element (TRE) to derive doxycycline (dox) inducible cell lines. In the presence of dox, the rtTA binds to the TRE, causing transcriptional activation of the downstream transgene. hESC were co-cultured on murine OP-9 stromal cell layers with thrombopoietin to promote megakaryocytic differentiation. For construction of the inducible mESC lines, mESCs already containing the rtTA at the ROSA26 locus were electroporated with either murine MKL1 (mMKL1) or murine RBM15-MKL1 (mRM). Site-specific insertion of the transgenes using a cre recombinase and modified loxP sites placed mMKL1 or mRM under the control of a TRE. The inducible mMKL1 mESCs were differentiated into hematopoietic cells using embryoid body formation and subsequently co-cultured on OP-9s. The human and murine MKL1 and RM constructs were transiently transfected into 293FT cells and Western blotting was used to confirm their functionality. In the hESC studies, GFP expression was detected by flow cytometry 24 hrs after dox induction in both cell lines, with 54.7% (SD+3.1) GFP+ cells in hMKL1 transduced cells and 47.5% (SD+16.2) GFP+ cells in hRM transduced cells. hMKL1 protein was detected 24 hrs after dox induction; however, the hRM fusion protein was not detected in undifferentiated hESCs by Western blot or IP/IB after 6, 24, 48, 72, 96 hrs of dox exposure. Nearly one fourth of the cells transduced with hRM were GFP+ after only 6 hrs of doxycycline exposure. Interestingly, with continued dox exposure over six days, GFP expression in the undifferentiated hESCs declined, with a mean decrease in GFP expression of 14.5% in both cell lines. Successful incorporation of the vector carrying the hRM was confirmed by genomic PCR and resulted in dox-inducible expression of hRM mRNA detected by RT-PCR. Upon megakaryocytic differentiation, GFP and CD41 expression, detected by flow cytometry, occur in a mutually exclusive fashion in both MKL and RBM15-MKL1 transduced hESCs. In the mESC experiements, protein expression of mMKL1 in the undifferentiated cells was confirmed by Western blot 24, 48, and 72 hrs after dox addition. Expression of mRM protein in the undifferentiated mESCs was undetectable 6, 14, 24, 30, 48, or 72 hrs after dox addition. However, mRM mRNA was detected by qRT-PCR at 0, 6, 14, and 30 hours after dox treatment in the undifferentiated mESCs. Of note, transient transfection of undifferentiated mESCs with mRM resulted in protein expression by Western blot as early as 6 hrs after transfection. The fusion protein continued to be detected at 12 and 18 hrs after transfection, but by 24 hrs, it was no longer detectable. When differentiated, the mMKL1 mESC line no longer demonstrated inducible mMKL1 expression in the CD41+ (hematopoietic) population as determined by qRT-PCR. These findings confirm ongoing activity of the promoters in the undifferentiated hESC and mESC lines and indicate that the promoters may be silenced upon induction of hematopoietic differentiation of both hESC and mESC lines. In addition, the transient detection of RM after transfection into the mESCs suggests post-translational modification of the fusion protein likely results in its rapid degradation. Disclosures: No relevant conflicts of interest to declare.

Differentiation of Human Natural Killer Cells From Pluripotent Stem Cells In a Feeder Free System Amenable to Clinical Translation

Abstract 107 Human natural killer (NK) cells are an attractive source of lymphocytes for adoptive immunotherapy. Although our understanding of natural killer cell biology continually grows, translating these concepts to the clinic has fallen behind. Currently, NK cell adoptive immunotherapy is only beneficial to a limited number of patients, particularly those with acute myelogenous leukemia (AML). In order to generate NK cells that are effective against a broader range of malignancies, our lab has focused on the generation of NK cells from human pluripotent stem cells. We have previously demonstrated the potency of NK cells derived from human embryonic stem cells (hESCs) both in vitro and in vivo. These previous studies utilized a stromal cell co-culture system to derive hematopoietic progenitor cells (CD34+CD45+ cells) from hESCs that can then produce CD45+CD56+ NK cells in a secondary culture system. More recently, we used a similar co-culture system to generate fully functional NK cells from several human induced pluripotent stem cell (iPSC) lines, which could provide NK cells on a patient-specific basis. Both hESC and iPSC-derived NK cells consist of a mature, homogenous population of cells expressing CD56, CD94, killer immunoglobulin-like receptors (KIRs), CD16, and the apoptosis-inducing ligands FasL and TRAIL. We now aim to convert this system into a completely defined stromal cell-free system. These studies will make this system more amenable to clinical scale up and allow us to better define elements essential for hematopoietic and NK cell development from human pluripotent stem cells. For example, hESC-derived NK cells would require minimal cell processing compared peripheral blood NK (PB-NK) cells that typically requires depletion of CD3+ and CD20+ cells to prevent GVH and passenger lymphocyte syndrome, respectively. Neither T cells nor B cells are present in our cultures, preventing these complications. Here, we have now used a spin embryoid body (“spin-EB”) approach potentially suitable for clinical scale-up. In these spin-EB cultures, defined numbers of undifferentiated hESCs or iPSCs are first aggregated in 96 well plates by centrifugation (3000 cells per well) in serum-free media containing only the cytokines SCF, BMP4, and VEGF. Under these stage 1 conditions, hematopoietic progenitor cells that express CD34, CD45, CD43, and CD31 develop and expand over 6–11 days. After this time, EBs are directly transferred (without dissociation or sorting) to Stage 2 cultures with EL08 stromal cells or stroma-free conditions in serum-free media containing NK cell-initiating cytokines (IL15, SCF, FLT3L, IL7, and IL3). We find that these Stage 2 cells acquire all the typical markers of mature NK cells (CD56, CD94, KIRs, etc). They also kill the CML target K562 cells at similar effector-to-target ratios as stromal-derived NK cells and PB-NK cells. Most notably, spin-EB-derived NK cells generated in entirely feeder free conditions also exhibit a typical NK cell phenotype and kill K562 targets. However, the NK cells derived in feeder-free Stage 2 conditions are less proliferative than those cultured in Stage 2 conditions on EL08 cells. Specifically, 12,000 undifferentiated hESCs or iPSCs give rise to 0.8–1 × 106 NK cells using EL08 in Stage 2 cultures compared to 0.4– 1 × 105 cells using the feeder-free Stage 2 conditions. Additionally, the efficiency of NK cell development from spin-EB cultures is not uniform: 58% of EBs produce NK cells in Stage 2 using EL08 stromal cells, and 25% of EBs produce NK cells in the completely feeder-free system. Based on these calculations, given 100% efficiency of this spin-EB NK cell developmental system, we estimate that 240,000 starting hESCs would provide enough NK cells to treat a single patient with a dose of 20 × 106 NK cells. Even at the lower efficiencies demonstrated here, one plate of undifferentiated hESCs or iPSCs (typically with 5–10 × 106 undifferentiated cells) could treat several patients. Ongoing work is focused on enhancing the percentage of hematopoietic progenitors obtained from the spin EB approach and expanding feeder-free NK cells with artificial antigen presenting cells in Stage 2 conditions. Disclosures: No relevant conflicts of interest to declare.

Mechanical Characterization of Differentiated Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) possess an immense potential in a variety of regenerative applications. A firm understanding of hESC mechanics, on the single cell level, may provide great insight into the role of biophysical forces in the maintenance of cellular phenotype and elucidate mechanical cues promoting differentiation along various mesenchymal lineages. Moreover, cellular biomechanics can provide an additional tool for characterizing stem cells as they follow certain differentiation lineages, and thus may aid in identifying differentiated hESCs, which are most suitable for tissue engineering. This study examined the viscoelastic properties of single undifferentiated hESCs, chondrogenically differentiated hESC subpopulations, mesenchymal stem cells (MSCs), and articular chondrocytes (ACs). hESC chondrogenesis was induced using either transforming growth factor-β1(TGF-β1) or knock out serum replacer as differentiation agents, and the resulting cell populations were separated based on density. All cell groups were mechanically tested using unconfined creep cytocompression. Analyses of subpopulations from all differentiation regimens resulted in a spectrum of mechanical and morphological properties spanning the range of hESCs to MSCs to ACs. Density separation was further successful in isolating cellular subpopulations with distinct mechanical properties. The instantaneous and relaxed moduli of subpopulations from TGF-β1 differentiation regimen were statistically greater than those of undifferentiated hESCs. In addition, two subpopulations from the TGF-β1 group were identified, which were not statistically different from native articular chondrocytes in their instantaneous and relaxed moduli, as well as their apparent viscosity. Identification of a differentiated hESC subpopulation with similar mechanical properties as native chondrocytes may provide an excellent cell source for tissue engineering applications. These cells will need to withstand any mechanical stimulation regimen employed to augment the mechanical and biochemical characteristics of the neotissue. Density separation was effective at purifying distinct populations of cells. A differentiated hESC subpopulation was identified with both similar mechanical and morphological characteristics as ACs. Future research may utilize this cell source in cartilage regeneration efforts.

Derivation of SSEA4-/CD73+ Mesenchymal Stem Cells from Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) could potentially provide a renewable source of different types of cells for cell therapy applications. Recently, mesenchymal stem cells (MSCs) have been derived from hESCs either through co-culturing with murine OP9 bone marrow stromal cell line or directly from hESCs without co-culturing with OP9 cells. Although the latter methodology is clinically advantageous over co-culturing with an animal cell layer those mesenchymal cells were reported to be positive for SSEA4. SSEA4 is a marker of undifferentiated hESCs and thus the presence of this marker on hESC-derived cells could potentially be problematic for clinical applications. We have recently achieved a novel and reproducible methodology for deriving a pure population of SSEA4-/CD73+ MSCs from federally approved hESC lines H1 and H9. To initiate the differentiation of hESCs to MSCs, we cultured undifferentiated hESCs on matrigel plates in murine embryonic fibroblast conditioned media with media changes every 3 days. Under these culture conditions a portion of embryonic stem cells differentiated into fibroblast looking cells. Through a multi-step process which involved the use of a culture methodology similar to what is being used to culture bone marrow (BM)-derived MSCs, and passaging cultured cells at defined time points we were able to derive a pure population of cells that were uniformly positive for MSC marker CD73 in about a 4-weeks period. These cells had fibroblast/mesenchymal looking morphology, and expressed cell surface marker antigens similar to what has been reported for adult human BM-derived MSCs: they are positive for CD29, CD44, CD54, CD71, CD90, glycophorin A, CD105, and were negative for hematopoietic markers such as CD34 and CD45. Similar to adult BM-derived MSCs these cells express HLA class-I antigens but not class-II antigens. Using established differentiation protocols we could differentiate the hESC-derived CD73+ MSCs into adipocytes, osteocytes, and chondrocytes as verified by immunohistochemistry and RT-PCR assays. So far we have grown these CD73+ MSCs up to passages 15–18. These cells retained their differentiation potential, and were karotypically normal when tested at passage 12. Most importantly, we did not observe any MSCs that were double positive for CD73 and SSEA4 antigen at any time point during our experiments. MSCs from a variety of fetal and adult sources are in various stages of clinical trials with some encouraging preliminary results. Our hESC-derived MSCs that are very similar to adult BM-derived MSCs regarding their growth and morphologic properties, immunophenotypic characteristics, differentiation potential, and importantly are devoid of hESC marker SSEA4 could potentially provide a novel source of MSCs for clinical applications.