Optimized media and workflow for the expansion of human pluripotent stem cells as aggregates in suspension cultures

3D suspension culture enables scale-up of human pluripotent stem cell (hPSC) manufacturing. However, media and methods optimized for 2D adherent cultures can lead to low volumetric productivity and laborious workflow in suspension cultures. To overcome these limitations we developed fed-batch media based on either mTeSRTM1 (BSA-containing) or TeSRTM-E8TM (animal component-free) for hPSC expansion as aggregates in suspension cultures. Fed-batch feeding protocols are more efficient and cost-effective than batch media changes because only exhausted components are replenished. Optimization studies were performed using human embryonic (H7 and H9), and human induced pluripotent (WLS-1C and STiPS-M001) stem cell lines. Suspension cultures were fed daily using either 50% medium exchanges of standard 2D media, or fed-batch optimized media and protocols. hPSC aggregate diameter must be kept below 350 μm to maintain cell viability and phenotype. With observed growth rates, aggregates required passaging every 3 or 4 days into clumps of 5-10 cells with Gentle Cell Dissociation Reagent. Clumps were re-seeded into fresh test medium plus 10 μM Y-27632. Passaging and feeding cycles were repeated for at least 5 passages. Optimization was performed by iteratively modifying the feed solution to maintain consistent nutrient levels and maximal growth rate while maintaining cell quality. Control and optimized fed-batch formulations demonstrated between 1.4 and 1.8-fold expansion per day, \u3e90% viability, Oct4 and TRA-1-60 expression \u3e90%, in vitro trilineage differentiation, and normal karyotype (n=8 independent cultures). Suspension culture optimized mTeSRTM-3D or TeSRTM-E8TM3D fed-batch media enables the cost-effective production of hPSCs as aggregates with efficient workflow and high cell quality

Optimized media and workflow for the expansion of human pluripotent stem cells as aggregates in suspension

3D suspension culture enables the efficient and cost-effective scale-up of human pluripotent stem cell (hPSCs) manufacturing. However, media optimized for 2D adherent cultures can lead to low volumetric productivity and inefficient workflow. To overcome these limitations we developed mTeSRTM3D, a defined medium based on mTeSRTM1, and novel protocols for fed-batch culture of hPSC aggregates. Human embryonic stem cell (hESC) lines (H1 or H9) or human induced pluripotent stem cell (hiPSC) lines (WLS-1C or STiPS-M001) that were previously maintained in 2D mTeSRTM1 culture were seeded into multiple suspension culture vessels containing mTeSRTM3D Seed Medium plus 10 μM Y-27632 ROCK inhibitor. 3D cultures were maintained using either daily 50% mTeSRTM1 medium exchanges (control) or using a fed-batch protocol whereby the culture medium was supplemented daily with mTeSRTM3D Feed Medium. After 3 or 4 days in suspension culture, aggregates were harvested, dissociated into small clumps with Gentle Cell Dissociation Reagent (GCDR) or single cell suspensions enzymatically, and re-seeded in mTeSRTM3D Seed Medium plus 10 μM Y-27632. Passaging and feeding cycles were repeated for at least 5 passages. 3D cultures were assessed for growth, viability, hPSC marker expression, in vitro differentiation potential, and karyotype. In addition, media was analyzed for molar glucose to lactate yield to characterize metabolism. By day 4, aggregates cultured in mTeSRTM3D typically grew to a mean diameter of 350 μm, with a 5-fold increase in cell number. Using mTeSRTM3D up to 109 cells can be produced from a single plate within 2-3 weeks representing a greater than 500-fold expansion. hPSC cultures maintained in mTeSRTM3D differentiated into all 3 germ layers with high efficiency. The average volumetric productivities were 0.7, 3.1 and 6.9 (x105) viable cells / mL in 2D, daily 50% media exchange, and mTeSRTM3D cultures, respectively. Using the GCDR clump passaging protocol, mTeSRTM3D cultured hPSCs retained normal karyotypes. Culture performance was evaluated in shaker bottles, spinner flasks and bioreactors. Performance in each culture system was comparable confirming straightforward scale-up and wide applicability. Typical growth rates were on the order of 1.5-fold expansion per day. Metabolic activity as assessed by the moles lactate produced to glucose consumed was 1.7, consistent with a primarily glycolytic metabolism. Image analysis was performed to estimate aggregate size during growth. Adaptation times for cells moving from 2D to 3D aggregate culture varied with different cell lines; typically one passage in 3D was required before consistent expansion passage over passage was obtained. Additionally, protocols were developed for use on a Hamilton® robotic platform for reproducible, matrix-free, high-throughput hPSC suspension culture at a small scale. mTeSRTM3D enables efficient scale-up and scale-down of hPSC cultures with greatly simplified workflow

Characterizing the effects of N/NRTIs on human telomerase activity in vitro and telomere maintenance in a transformed human cell model

Telomeres are nucleoprotein structures found at the ends of most linear chromosomes. Telomeric DNA shortens with each cell division, effectively restricting the proliferative capacity of most human cells. Telomerase, a specialized reverse transcriptase (RT), is responsible for de novo synthesis of telomeric DNA, and is the only physiological mechanism through which some human cells extend their telomere length. Disruption in telomerase activity results in accelerated telomere attrition, which manifests as a loss in tissue regenerative capacity. In individuals infected with the human immunodeficiency virus (HIV), current clinical treatment guidelines prescribe the use of a long-term, combination drug therapy known as highly active anti-retroviral therapy (HAART). Nucleoside and non-nucleoside reverse transcriptase inhibitors (N/NRTIs) inhibit HIV RT and are integral components of HAART. There are both reported structural and mechanistic similarities between telomerase RT and HIV RT. Based on these observations, we hypothesized that N/NRTIs will inhibit telomerase in the same ways that they inhibit HIV RT, and that long-term exposure to these agents will limit telomere maintenance in telomerase-dependent cells. We tested our hypothesis using two approaches. First, N/NRTIs were tested against telomerase activity in vitro using a primer extension assay. All NRTIs tested in this assay inhibited human telomerase, and their relative potencies were compared to their respective dideoxynucleotide analog counterparts. The NNRTIs, which are non-competitive inhibitors of HIV RT, did not inhibit telomerase. In our second approach, we tested the effects of long-term, continuous treatment with N/NRTIs on telomere length maintenance in a transformed human cell model with constitutive telomerase activity. The rates of telomere length attrition in the presence of high doses of several NRTIs were consistent with maximal telomerase inhibition. In contrast, I observed minimal effects on telomere maintenance in cells treated with NNRTIs. My primer extension assay data corroborate conclusions from previous studies on telomerase biochemistry and support mechanistic conservation between telomerase RT and HIV RT. Collectively, my biochemical and cell culture studies demonstrated that telomerase inhibition by NRTIs could potentially lead to treatment complications in current antiretroviral therapies and encourage large-scale clinical and epidemiological studies on the effects of telomerase inhibition by these drugs.Medicine, Faculty ofMedical Genetics, Department ofGraduat

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

<div><p>Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.</p> </div

Summary of the relative potencies of selected NRTIs against telomerase catalysis <i>in vitro.</i>

*<p>Discrimination Factor (DF): fold increase in concentration of ddNTPs and NRTIs above respective endogenous competitor dNTP required for 50% telomerase inhibition <i>in vitro</i> (IC₅₀).</p><p>[DF = (IC50_NRTI/ddNTP)÷([dNTP_competitor])].</p

Optimization of the telomerase primer extension assay.

<p><b>A.</b> Optimization of the primer extension assay for telomerase activity. Sources of telomerase activity: lane 1, whole cell lysate from 293HEK cells stably transfected with TERT and TER; lane 2, whole cell lysate from 293HEK cells transiently transfected with TERT and TER; lane 3, IP telomerase from 293HEK cells transiently transfected with 3×FLAG TERT and TER. Brackets in lane 2 denote DNA degraded by endogenous nucleases. <b>B.</b> IP telomerase activity in the presence of six different primers demonstrating that the primer extension assay is telomerase-specific. The DNA banding pattern with each primer can be predicted based on the template domain in TER. The number of nt added to each primer after first-repeat synthesis is shown below each lane.</p

Continuous treatment of HT29 cells with the adenosine analogs TDF and ddI causes observable telomere shortening. A.

<p>TRF blots of untreated (left), TDF-treated (right) HT29 cells. PDL at which TRF was analyzed is shown above each lane. Molecular mass markers are shown at left and right of gel images. Each TRF smear was quantified as a weighted average and is shown below each lane. <b>B.</b> Growth curves and telomere maintenance dynamics of HT29 cells treated continuously with TDF. The growth curve and TRF dynamics of untreated HT29 cells (solid blue line) is plotted for comparison. There were moderate levels of telomere length loss in both the lower (50 µM) and higher (100 µM) TDF doses. However, these observations are marred by TDF cellular toxicities that prevent longer-term TRF analysis. <b>C.</b> TRF blots of DMSO-treated (control vehicle, left) and ddI-treated (right) HT29 cells. <b>D.</b> Growth curves and telomere maintenance dynamics of HT29 cells treated continuously with ddI. The growth curve and TRF dynamics of DMSO-treated HT29 cells (solid blue line) is plotted for comparison. Moderate telomere length loss over time was observed in all three doses (30 µM, 60 µM and 120 µM) of ddI treatments.</p

Summary of the impact of NRTIs/NNRTIs on telomere maintenance in HT29 colorectal adenocarcinoma cells.

<p>Summary of the impact of NRTIs/NNRTIs on telomere maintenance in HT29 colorectal adenocarcinoma cells.</p

The NNRTIs NVP and EFV do not inhibit telomerase <i>in vitro</i>.

<p>Representative gel images showing telomerase activity in the presence of either 1 mM (<b>A</b>) or 4 mM (<b>B</b>) NVP or EFV. 5% DMSO was used as a vehicle control. <b>C.</b> Quantification of gel image shown in A. Data was obtained through the analysis of at least three independent experiments.</p

The guanosine analog CBV-TP inhibits telomerase <i>in vitro</i>. A.

<p>In the presence of increasing concentrations of ddGTP or CBV-TP (shown as G*), the radioactive signal at the primer +3 position diminishes. <b>B.</b> Representative gel images of IP telomerase activity in the presence of ddGTP or CBV-TP. Telomerase-specific DNA products are labeled on the left and right of each gel. Free, 18-nt end-labeled primer is shown for reference. <b>C.</b> Dose-response curves demonstrating telomerase inhibition by guanosine analogs. Solid line, ddGTP; dashed line, CBV-TP. Data were obtained from a minimum of three independent experiments per guanosine analog. Error bars are mean ± SD. RC denotes recovery control.</p