Reducing variability in conditions for cell handling improves MSC yields

Efficient cell expansion in vitro is essential to commercialization of human MSC as a cellular therapy. The cost of goods sold (COGS) is dramatically affected by how long it takes to expand the cells in vitro and the cell yield determines the number of doses generated for profit. Therefore, maximizing MSC growth in culture is critical for the success of MSC-based cellular therapies. Studies by others have shown that temperature differences in cell production can adversely affect cell yields. Here we study the effects of variability in temperature and CO2, like changes seen during routine cell handling in a room air BSC, on human MSC yield. We cultured human bone marrow mesenchymal stromal/stem cells for 8 biweekly subpassages (P4-P12) with conventional room air CO2 incubator conditions (37 degrees C/ 5% CO2). The culture was divided into separate cultures for routine cell handling in two different conditions (1) room air BSC conditions (RT/ 0.1% CO2) (variable) or (2) the same conditions as incubation (constant). At each passage, cells were plated in 96-well plates which were assayed over time for cell growth kinetics. Consistently, MSC incubated and handled in constant conditions recovered more quickly after subpassage and were more likely to continue to divide, improving final cell yields. We conclude that constant conditions for cell handling are critical for maximum MSC cell yield

Scale-out of massively parallel patient-specific cell cultures with a modified transportable conditioned cell culture chamber

Barrier Isolators, which separate the cell culture processing atmosphere from the bioburden of personnel, are the best means to reduce contamination risks. These isolators are currently being used for cGMP-compliant clinical trials1, 2. Scaling cell production processes presents non-obvious restrictions to most people. Compared to open processing, modular Cytocentric isolators can be replicated to scale proportionately with each stage in cell processing until all steps are accommodated maximally. This allows a process to efficiently and quickly scale with operations from pre-clinical through clinical studies3. However, for processing of massively parallel patient-specific cell cultures, incubation capacity in a barrier isolator, unlike in the open room, can be a bottleneck. Inexpensive and infinitely elastic incubation capacity can be provided by existing external incubators if cultures can be safely transported to and from the isolator for processing. We tested a modified transportable conditioned cell culture chamber (TC4) designed to enclose cell cultures inside the exterior incubator and fit through the airlocks of the barrier isolator to safely deliver cells to the interior for processing. We have previously published on good cell growth using this processing system to expand K562 cells, a hematopoietic stem cell-like cell line that has been used as a surrogate for CAR-T cell processing. In this study, we addressed sterility concerns by running mock production runs with a highly permissive color-changing bacterial broth. We ran three production runs, moving mock cultures between the barrier isolator and the external incubator with the TC4 transport chamber. We took samples of the final mock cell product, sealed them into sterile vials, and incubated them long-term, monitoring for bacterial growth. We also performed environmental monitoring of the barrier isolator processing chamber with an air sampler and contact plates. Positive control samples were all yellow and turbid. Negative samples and all test materials were negative for microbial growth. We concluded that this transport chamber could help safely alleviate the bottleneck in cell production presented by the unique needs of massively-parallel patient specific cell incubation. References: Mei, S.H., et al., Isolation and large-scale expansion of bone marrow-derived mesenchymal stem cells with serum-free media under GMP-compliance. mortality, 2014. 40: p. 1. Marathe, C.S., et al., Islet cell transplantation in Australia: screening, remote transplantation, and incretin hormone secretion in insulin independent patients. Horm Metab Res, 2015. 47(1): p. 16-23. Yufit, T., P. Carson, and V. Falanga, Topical Delivery of Cultured Stem Cells to Human Non-Healing Wounds: GMP Facility Development in an Academic Setting and FDA Requirements for an IND and Human Testing. Current drug delivery, 2014. 11(5): p. 572-581

Cytocentric measurement for regenerative medicine

Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions can be controlled to meet the needs of cells and tissues in culture for RM, we have to know what those needs are and be able to quantify them. Therefore, identification and measurement of critical cell quality attributes at a cellular or pericellular level is essential to generating reproducible cell and tissue products. Here, we identify some of the critical cell and process parameters for cell and tissue products as well as technologies available for sensing them. We also discuss available and needed technologies for monitoring both 2D and 3D cultures to manufacture reliable cell and tissue products for clinical and non-clinical use. As any industry matures, it improves and standardizes the quality of its products. Cytocentric measurement of cell and tissue quality attributes are needed for RM

Fine-Scale Human Population Structure in Southern Africa Reflects Ecogeographic Boundaries.

Recent genetic studies have established that the KhoeSan populations of southern Africa are distinct from all other African populations and have remained largely isolated during human prehistory until ∼2000 years ago. Dozens of different KhoeSan groups exist, belonging to three different language families, but very little is known about their population history. We examine new genome-wide polymorphism data and whole mitochondrial genomes for >100 South Africans from the ≠Khomani San and Nama populations of the Northern Cape, analyzed in conjunction with 19 additional southern African populations. Our analyses reveal fine-scale population structure in and around the Kalahari Desert. Surprisingly, this structure does not always correspond to linguistic or subsistence categories as previously suggested, but rather reflects the role of geographic barriers and the ecology of the greater Kalahari Basin. Regardless of subsistence strategy, the indigenous Khoe-speaking Nama pastoralists and the N|u-speaking ≠Khomani (formerly hunter-gatherers) share ancestry with other Khoe-speaking forager populations that form a rim around the Kalahari Desert. We reconstruct earlier migration patterns and estimate that the southern Kalahari populations were among the last to experience gene flow from Bantu speakers, ∼14 generations ago. We conclude that local adoption of pastoralism, at least by the Nama, appears to have been primarily a cultural process with limited genetic impact from eastern Africa

Fine-Scale Human Population Structure in Southern Africa Reflects Ecogeographic Boundaries

Recent genetic studies have established that the KhoeSan populations of southern Africa are distinct from all other African populations and have remained largely isolated during human prehistory until ∼2000 years ago. Dozens of different KhoeSan groups exist, belonging to three different language families, but very little is known about their population history. We examine new genome-wide polymorphism data and whole mitochondrial genomes for >100 South Africans from the ≠Khomani San and Nama populations of the Northern Cape, analyzed in conjunction with 19 additional southern African populations. Our analyses reveal fine-scale population structure in and around the Kalahari Desert. Surprisingly, this structure does not always correspond to linguistic or subsistence categories as previously suggested, but rather reflects the role of geographic barriers and the ecology of the greater Kalahari Basin. Regardless of subsistence strategy, the indigenous Khoe-speaking Nama pastoralists and the N|u-speaking ≠Khomani (formerly hunter-gatherers) share ancestry with other Khoe-speaking forager populations that form a rim around the Kalahari Desert. We reconstruct earlier migration patterns and estimate that the southern Kalahari populations were among the last to experience gene flow from Bantu speakers, ∼14 generations ago. We conclude that local adoption of pastoralism, at least by the Nama, appears to have been primarily a cultural process with limited genetic impact from eastern Africa

Rapid evolution of a skin-lightening allele in southern African KhoeSan.

Skin pigmentation is under strong directional selection in northern European and Asian populations. The indigenous KhoeSan populations of far southern Africa have lighter skin than other sub-Saharan African populations, potentially reflecting local adaptation to a region of Africa with reduced UV radiation. Here, we demonstrate that a canonical Eurasian skin pigmentation gene, SLC24A5, was introduced to southern Africa via recent migration and experienced strong adaptive evolution in the KhoeSan. To reconstruct the evolution of skin pigmentation, we collected phenotypes from over 400 ≠Khomani San and Nama individuals and high-throughput sequenced candidate pigmentation genes. The derived causal allele in SLC24A5, p.Ala111Thr, significantly lightens basal skin pigmentation in the KhoeSan and explains 8 to 15% of phenotypic variance in these populations. The frequency of this allele (33 to 53%) is far greater than expected from colonial period European gene flow; however, the most common derived haplotype is identical among European, eastern African, and KhoeSan individuals. Using four-population demographic simulations with selection, we show that the allele was introduced into the KhoeSan only 2,000 y ago via a back-to-Africa migration and then experienced a selective sweep (s = 0.04 to 0.05 in ≠Khomani and Nama). The SLC24A5 locus is both a rare example of intense, ongoing adaptation in very recent human history, as well as an adaptive gene flow at a pigmentation locus in humans

Diversity in Compartmental Dynamics of Gene Regulatory Networks: The Immune Response in Primary Influenza A Infection in Mice

<div><p>Current approaches to study transcriptional profiles post influenza infection typically rely on tissue sampling from one or two sites at a few time points, such as spleen and lung in murine models. In this study, we infected female C57/BL6 mice intranasally with mouse-adapted H3N2/Hong Kong/X31 avian influenza A virus, and then analyzed the gene expression profiles in four different compartments (blood, lung, mediastinal lymph nodes, and spleen) over 11 consecutive days post infection. These data were analyzed by an advanced statistical procedure based on ordinary differential equation (ODE) modeling. Vastly different lists of significant genes were identified by the same statistical procedure in each compartment. Only 11 of them are significant in all four compartments. We classified significant genes in each compartment into co-expressed modules based on temporal expression patterns. We then performed functional enrichment analysis on these co-expression modules and identified significant pathway and functional motifs. Finally, we used an ODE based model to reconstruct gene regulatory network (GRN) for each compartment and studied their network properties.</p></div

Distance from sub-Saharan Africa predicts mutational load in diverse human genomes.

The Out-of-Africa (OOA) dispersal ∼ 50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive