ArunA Biomedical, Inc.

The Good, the Bad and the Ugly of hPSC Culture (Part I: Feeder Layer System)

2011-10-04T15:41:00.000-04:00

By: Jamie Chilton, PhD.

When conversations turn to work, I laughingly tell my friends that my stem cell cultures are, in fact, harder to take care of than a toddler. Ok, so I admit I’m the only one that happens to find that joke amusing. But I am sure you stem cell biologists out there can empathize when I say culturing human pluripotent stem cells (hPSC) is no mean feat, whether you are talking about induced pluripotent stem cells (hiPSC) or embryonic stem cells (hESC).

When I started culturing human pluripotent stem cells (hPSC), I wish there had been some easily accessible checklist to make sure I was on the right track, instead of relying on time-consuming literature searches. Not all of us may be lucky enough to follow in the footsteps of a graduate student, post doc or researcher who has worked out all the kinks for us.

So I decided to put together a cheat sheet to help the beginner recognize and maintain hPSC. Part I will be included in this blog post and describe hPSC cultured on a feeder layer of mouse embryonic fibroblasts (MEFs).

Stay tuned for a future blog post which will contain Part II describing feeder-free hPSC cultures.

Quick Reminders on Culturing hPSC on MEFs:

Maintaining hPSC in a pluripotent state and preventing them from differentiating requires rigorous cell culture efforts and continual dedication.
Every day, observe your hPSC colonies closely and be very selective about choosing high quality colonies for continued expansion.
Refresh the medium daily at the same time of day.
Keep complete growth medium formulations consistent, use fresh reagents to make complete growth medium.
Use fresh MEF feeder layers less than a week old.
Passage hPSC every 3-4 days before colonies start spontaneously differentiating.
Pick specific colonies with the best pluripotent morphology for continued propagation.

The images depicted below will show hPSC colonies with the proper morphology indicating a pluripotent state, as well as show differentiation of hPSC which should be avoided. The following images are female H9 (WA09) hESC (originally from WiCell Research Institute) cultured on MEFs, yet the concepts explained are applicable to hiPSC cultured on MEFs as well.

THE GOOD

MorphologicalCharacteristics of Human Pluripotent Stem Cells

Morphological characteristics to look forin human pluripotent stemcell (hPSC)colonies cultured on MEFs and maintaining a pluripotent state include:

1. Shape of colonies: truly pluripotent colonies will be very spherical in shape, not irregular. The rounder the better.

2. Colony definition: pluripotent colonies will be immediately distinguishable from the surrounding MEFs with well-defined borders.

3. Individual cell properties:

individual cells within a pluripotent colony will be irregular in shape with well-defined borders (either white or black depending on magnification and refraction);
individual cells will also have a high nuclear to cytoplasmic ratio.

4. Architecture: pluripotent colonies will have more of a 3D multi-layer architecture and will not be a single monolayer of cells.

5. Color: pluripotent colonies will appear “whitish” in color against the surrounding MEFs, with a very translucent, light-refractive appearance.

Tips

Pluripotent hPSC on MEFs should continue to proliferate readily with an approximate doubling time of 24 – 36 hours. Thus, hPSC colonies will need to be passaged every 3-4 days.
When manually passaging hPSC colonies off MEFs, they should come apart in “sticky” pieces.

THE BAD
Morphological Indications of Differentiation in Your Culture

1. It is normal for some colonies to differentiate spontaneously in hPSC culture. It can be interpreted as a healthy sign that the culture has the capacity to differentiate into multiple cell types. However, 20% or less of the culture should be spontaneously differentiating. If the majority of the colonies spontaneously differentiate, culture technique and conditions need to be reevaluated to determine what is causing the differentiation.

2. Morphologies to avoid:

Flat, monolayer colonies grayish in color.
Loss of refractive properties.

Overgrowth and overcrowding of colony-like structures.

Loose growth of cells on feeders, non colony-like structures.
Irregularly-shaped colony-like structure.

Colony-like structure overgrowing 3-dimensionally with beginnings of a necrotic core or resemblance to a differentiating embryoid body.

THE UGLY
You Don’t Want This Happening to Your Culture!

Resources for Guiding Stem Cell Based Assay Development

2011-09-19T09:45:00.000-04:00

By: Jessica McCabe,and Allan Powe, Jr., PhD.

With the number of FDA approved drugs per annum dwindling, the pharmaceutical industry is looking for new ways to increase the discovery rate of safe, clinically efficacious compounds at the very earliest stages of drug development. Currently, many of the cell lines used in early drug discovery efforts are highly engineered and often poorly reflect the native pharmacology of intended drug targets. One approach to improving this situation is to use human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) to generate various differentiated cell types of interest, e.g., dopaminergic or motor neurons. These hESC- and hiPSC-derived cells provide physiologically relevant, unadulterated in vitro cellular models that more closely reflect the biology and pharmacology of the corresponding cells in vivo. Better cellular models, could increase the probability of identifying more efficacious, safer drug candidates during hit-to-lead and lead optimization campaigns.

To help meet this need for improved, physiologically relevant cellular models, we have developed bioassays and applications for our neural and mesenchymal stem cell products with an eye toward use in drug discovery. Adhering to standards set by the pharmaceutical industry is of utmost importance in order to provide maximum utility for our drug discovery related applications. So, we look to several different websites as resources and guides in our assay development efforts. These are some of our favorites:

Assay Guidance Manual – This is the main resource we use to guide in-house assay development. The website, originally started as a collaboration between the National Chemical Genomics Center (NCGC) and Eli Lilly & Co., is now under the auspices of the National Institutes of Health Center for Translational Therapeutics (NCTT). The Assay Guidance Manual covers an extremely broad range of assays – cell based assays, enzymatic activity, receptor binding, FLIPR™ and functional assays for GPCRs and ion channels, immunoassays (including ELISAs and Westerns), RNAi screening, etc. The site also provides information on basic assay optimization and validation. Newer sections under development include in vivo assay guidelines and a section on stem cells and regenerative medicine.
LabAutopedia – This wiki-styled site was created by the Society for Laboratory Automation and Screening (SLAS) to provide a collaborative knowledgebase of the latest laboratory technology and covers topics such as laboratory automation, assay detection technologies, compound collections, genomics and proteomics, and high throughput screening. This site nicely complements the Assay Guidance Manual by providing more in-depth information on specific applications.

In addition to these sites, there are a few blogs we read regularly that help us think more broadly about stem cell assay development and to keep up-to-date on advances in the stem cell field:

SciClips – This web based, “open innovation platform” has some insightful blog entries on the utility and development of cell based assays in drug discovery. One entry along with the associated comments provides an excellent discussion of the current pros and cons of stem cell based assays as drug discovery tools.

Stem Cell Assays – Founded by William Gunn and Alexey Bersenev, this site provides not only links to protocols for newly published stem cell based methods but also casts a critical eye on stem cell research in an effort to promote increased levels of rigor in the field.

Stem Cells Portal - This site sponsored by AlphaMed Press highlights the latest news and scientific publications in stem cell research from a wide variety of journals, such as Science, Nature, and Cell, as well as those from its own press. The news feed for the Portal’s Facebook page provides a convenient, almost real-time way to keep up with newly published stem cell related articles.

We hope that you find these resources just as useful as we do in conducting your stem cell related research.

Outside of the Lab...

2011-07-29T16:02:00.001-04:00

By: Kim Galland

Hello, Everyone! Things are bustling around the ArunA laboratory, and I thought I’d take some time to step out of the lab to see what is happening elsewhere in the world of stem cells. Sometimes, I get so caught up in development that I forget just how far stem cell research has come. The Shepherd Center, based in Atlanta, Ga, has begun stem cell therapy on a patient who severed their spinal cord in an accident. Progress to the clinical phase is a reminder of how far stem cell research has progressed, and should serve as validation for continued discovery of improved uses for stem cells. Click below for the ABC news spotlight on the first stem cell therapy patient.

ABC World News: Spinal Cord Injury Victim First to Undergo Embyronic Stem Cell Therapy at Shepherd Center

No application is worth pursuing, without quality and repeatability. . .

2011-07-21T13:20:00.004-04:00

By: Tracey Worthington Stice

Over the last decade the speed at which advancements in the field of stem cell research has been impressive given decreased funding and political posturing. For those of us who have watched this industry grow from its infancy, the pace of these advancements is not without concern. Most in the field have a valid concern over the quality and consistency of commercially available cells and the environment from which products are deployed to the marketplace. Successful discoveries based in part or in whole through the use of stem cells, whether in therapeutic development or drug discovery, will be under increased scrutiny from federal agencies in the coming years. As suppliers to the market, we have a responsibility to provide materials that meet internationally recognized quality standards whether certified or practiced.

In its early stage of commercial product development, ArunA established a Quality Assurance program enabling us to manufacture stem cell products that are repeatable and highly scalable. This validated manufacturing process allows us to manufacture and release human embryonic stem cell derived neural and mesenchymal progenitor cells in the billions all-the-while demonstrating consistency lot-to-lot and batch-to-batch including karyotypic stability.
When you talk with your stem cell supplier, I encourage you to ask about their manufacturing practices, their product validation process and their quality assurance program. Then, call us.

iPSC Mountain or Molehill? Time Will Tell...

2011-06-13T12:55:00.000-04:00

By: Jamie Chilton, PhD

Recently, the induced pluripotent stem cell (iPSC) field raised an eyebrow over the results from an immunogenicity study conducted by Yang Xu¹ and colleagues at the University of California, San Diego. Using a teratoma model system, Xu et al reportedly found that mouse iPSC cells demonstrated rejection, T cell infiltration or tissue damage and regression in mice of the same genetic background following syngeneic transplantation. By comparison, embryonic stem cells isolated from the same mouse line showed no evident immune rejection. These findings raise doubts as to the utility of employing iPSC as a future human cellular therapy for a wide range of conditions including spinal injury, diabetes, Parkinson’s and heart disease.
In ensuing discussions over the results of the iPSC immunogenicity study, many in the iPSC field expressed surprise by the news. After all, we’re only taking adult somatic cells out of their natural in vivo niche and introducing factors to force them to completely rewind their biological clocks to a naïve state. Let’s see….full epigenome reset...appropriate gene and protein expression…accurate response to new microenvironmental cues and directed differentiation…after weeks in vitro, total immune tolerance following transplantation. Boy, this task list is no sweat.

But seriously, should we really be surprised? Should we have realistically placed such high expectations that everything concerning iPSC would be smooth sailing? I think we should take a moment not to be premature and unnecessarily overreact to the study’s findings, instead remain equally receptive of negative results to form a complete picture of the iPSC mechanisms at work in light of future regenerative therapies. On an optimistic note, I find it preferable to encounter hurdles earlier rather than later, while the iPSC field is still in its infancy. However, I know it is human nature to want to run from potential negative implications like falling stock in a recession. But let’s wait to see what we scientists collectively and reproducibly discover in time, with future immunogenicity studies extending beyond the teratoma model, exploring human iPSC from multiple sources and utilizing ever more precise and sophisticated reprogramming methods.

Nevertheless, even if this immunogenicity molehill ascends more steeply than anticipated and iPSC transplantation proves untenable, that’s not to say iPSC won’t become a powerful, invaluable tool in the development of therapeutics. Whether it’s disease or developmental modeling, high throughput screening or transforming therapeutic discovery and validation processes to reduce and complement animal testing, there is all the more reason to continue iPSC research.

(1) Zhao, T., Zhang, Z., Rong, Z. and Xu, Y. Immunogenicity of induced pluripotent stem cells. Nature. 2011 May 13. [Epub ahead of print]

Techniques and tips for using Matrigel as an ECM

2011-05-17T11:23:00.002-04:00

By: Jessica McCabe

As the Production Team Leader at ArunA Biomedical, my duties include addressing the technical needs of customers. Some questions that consistently come up regard plate coating. When plating ArunA's hNP1™ Human Neural Progenitor Cells, and hN2™ cells, we recommend the use of Matrigel™ (BD Biosciences Cat #354234). For some laboratories, using Matrigel successfully can be tricky. To alleviate some of the frustration, I have included a list of helpful tips for first time users to become accustomed to working with the product. These are the steps we use in the lab at ArunA to make 1:200 Matrigel™ dilution, which we then use to coat our plates:

It is extremely important to keep the Matrigel™, and all components used to make the solution cold/on ice.
Thaw the stock bottle of Matrigel™ overnight at 4C. (You may also thaw it on ice while in the refrigerator; the ice helps to buffer temperature fluctuations and keep the Matrigel™ cold.)
While on ice, dilute the thawed Matrigel™ with an equal volume of cold DMEM (e.g., mix 10 ml thawed Matrigel™ with 10 ml DMEM)- this makes a 1:2 dilution. We typically make 1-3ml aliquots. Immediately put the aliquots in the -20C.
Before use, thaw a 1:2 Matrigel™ aliquot overnight at 4C until fully thawed (approximately 24hrs).
Use the 1:2 dilution to further dilute to 1:200 (e.g., to make 100 ml solution, add 1ml of 1:2 Matrigel™ into 99 ml of DMEM).
Coat the plates you will use fresh each time and let the Matrigel™ coated plate incubate at 4C or room temp in the hood for 1-3 hours.
Be sure to wash once the PBS containing calcium and magnesium.
Before using, you may want to consider checking the plate under the microscope for debris (see reference below). Never use a prepared Matrigel™ solution for longer than 2 weeks

Once I became familiar with how to use Matrigel™, it quickly became an effective and efficient method for plating both the hNP1™ and hN2™ cells.

Left, Poor Matrigel coating (note the high level of debris on the plate, little to no cell attachment).

Right, Successful Matrigel coating (little to no debris on the plate, high level of cell attachment).

Transplantation of hNP1 neural progenitors in animal models for ischemic stroke

2011-04-28T17:00:00.004-04:00

By: Allan Powe, Jr., PhD

Since arriving at ArunA nearly a year ago, I have been amazed at the remarkable versatility of hNP1™ Human Neural Progenitor Cells. My own work and that of many others have shown that these cells can be used for a wide variety of in vitro research applications, including assays for proliferation and neurogenesis, neuronal differentiation, and cell migration. While these types of applications are important, part of the promise that stem cells hold is their application for cell replacement based therapies. Even though several clinical trials for stem cell transplantation are currently underway or in development, researchers still need high quality, readily available sources of neural stem cells for early stage, proof-of-concept development of such therapies.

Recently, I came across some interesting studies using hNP1™ cells for proof-of-concept stem cell transplantation studies in models of ischemic stroke. Ischemic stroke is the death of brain tissue due to obstruction of cerebral blood vessels and is the third most common cause of death in the US (1). Currently, efforts are underway to develop effective stem cell based treatments that would promote recovery from ischemic stroke (2). To understand how stem cell based therapies for stroke might be operating, the Greenberg laboratory at the Buck Institute for Aging Research conducted a series of studies examining the effect of neural progenitor cell (NPC) transplantation in a rat model for ischemic stroke using hNP1™ cells as a source of NPCs (3,4,5).

In their first study, they showed that co-injection of hNP1™ cells with Matrigel directly into the cortical infarct cavity at 14 days post-ischemia could reduce infarct volume by ~50% (3). Moreover, rats injected with the hNP1™ /Matrigel mix exhibited improved performance in motor and cognitive tests at eight weeks post-transplantation (3). The second study demonstrated that both young (3-month old) and aged (24-month old) rats benefited from transplantation (4), the implication being that aged stroke patients may benefit from stem cell transplantation.

Perhaps most interestingly, their third study indicated that NPC transplantation appeared to increase neurogenesis only in the subventricular zone (SVZ) ipsilateral to the site of the infarct and injection; neurogensis in the subgranular zone (SGZ) and contralateral SVZ were unaffected (5). The authors suggest that the transplantation of NPCs may stimulate a program of endogenous neurogenesis in addition to directly replacing neurons lost during the ischemic episode. This idea is supported by two additional pieces of evidence. In a separate study, the Greenberg lab had shown that endogenous neurogenesis by itself does restore some of the neuronal loss from an ischemic episode, although far from completely (6). Moreover, the number of NPC cells injected cannot account completely for the recover seen (3). Thus, using the hNP1™ neural progenitor cells, these investigators have begun to elucidate the basic mechanisms that underlie stem cell based therapies for ischemic stroke.

These studies highlight the really remarkable versatility of the hNP1™ Human Neural Progenitor Cells. Moreover, they open up entire avenues of investigation for proof-of-concept stem cell transplantation using the hNP1™ cells not only in models of stroke but also in models of neurodegenerative disorders, such as ALS, Alzheimer's or Parkinson's. I can't wait to see what's next...

1) http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5619a2.htm
2) Lindvall O, Kokaia Z. Stem cells in human neurodegenerative disorders--time for clinical translation? J Clin Invest. 2010 Jan 4;120(1):29-40
3) Jin K, Xie L, Mao X, Greenberg MB, Moore A, Peng B, Greenberg RB, Greenberg DA. Effecto of human neural precursor cell transplantation on endogenous neurogenesis after focal cerebral ischemia in the rat. Brain Res. 2011 Feb 16;1374:56-62. PMID: 21167824
4) Jin K, Xie L, Mao X, Greenberg MB, Moore A, Peng B, Greenberg RB, Greenberg DA. Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats. Aging Cell. 2010 Dec;9(6):1076-83. PMID: 20883527
5) Jin K, Mao X Xie L, Galvan V, Lai B, Wang Y, Gorostiza O, Wang X, Greenberg DA. Transplantation of human neural precursor cells in Matrigel scaffolding improves outcomes from focal cerebral ischemia after delayed postischemic treatment in rats. J Cereb Blood Flow Metab. 2010 Mar;30:534-44. PMID: 19826433
6) Jin K, Wang X, Xie L Mao L, Mao XO, Greenberg DA, Transgenic ablation of doublecortin-expressing cells suppresses adjult neurogenesis and worsens stroke outcome in mice. Proc Natl Acad Sci USA 2010 Apr 27; 107(17)7993-8. PubMed PMID: 20385829

A State of Confluence and Neural Progenitors

2011-04-01T14:33:00.001-04:00

By: Kim Galland

When I started as a new ArunA employee, I had to adjust my previous notions of cell passaging when it came to culturing hNP1™ human neural progenitor cells. My past experience was in a laboratory that cultures bovine kidney cells (BK.) The BK cells are to be passaged when they reach 80%-95% confluency and they offer leniency in their state of confluence when passaged. I have passaged the kidney cells anywhere between 75% and 100% confluency. However, the maintenance and the passaging of human neural progenitor cells requires a little more finesse. It took me several passages to realize that "100% confluency" did not have wiggle room. Neural progenitor cells do not thrive when passaged at 80 or 90% confluence.

Neural progenitors are highly density dependent, but why? Studies suggest that neural progenitors are constantly signaling each other via short range mechanisms linked to purine receptors, which indicates a need for close contact. (Lin, Jane H.C. et al). Additionally, the use of growth factors in our proliferation media allows the cells to "cross talk", and the purine neucleotides function as proliferation signals for the neural progenitors. Therefore, if the cells are not at 100% confluence during passage, one runs the risk of the cells seating too far apart post passage. This may lead to a lack of proliferation and expansion and the cells may begin to differentiate.

Below, I've demonstrated key characteristics to look for when working with our hNP1™ Human Neural Progenitor Cells.

Citation from:
Purinergic signaling regulates neural progenitor cell expansion and neurogenesis. Lin, Jane H.C. et al, Dev. Biol. 2007 February 1; 302(1): 356-366

Quality Control for iPSC and ESC Derived Products

2011-02-21T15:30:00.004-05:00

By: Rebecca Kirkland

Drug discovery strategies are evolving to recognize the value of defeat -- the earlier the better. Drug candidates that fail Phase I clinical trials are most often rejected because of unanticipated toxicity. The cost to bring a promising new compound to the clinic is substantial and must be managed wisely. Characterization of new drug candidates begins with in vitro studies that can quickly reveal toxicity and unintended cellular responses, leading to elimination of unsatisfactory compounds at an early preclinical stage of drug discovery. A more robust early elimination process can preclude the expense of testing unsuitable drug candidates in clinical trials and improve the chances for ultimate approval of safe and effective drugs.

One of the major goals for stem cell research includes developing cell lines that closely mimic the cellular environment found in tissues affected by certain diseases. The closer cells in culture can recapitulate in vivo pathology, the better models these in vitro studies will be. At the moment, many cell lines representing various tissue and disease types are commercially available to test new drug candidates for safety and efficacy in vitro. However, stem cell lines must be monitored for chromosomal changes that can come to predominate in a culture if the genetic alteration provides a selective advantage in culture. Trisomies of chromosomes 12 and 17q are common abnormalities encountered by independent laboratories propagating stem cells^[1]. These and other subtle genetic flaws can be detected at an early stage with routine testing now.

ArunA Biomedical is committed to excellence in our products and services. Quality control testing is done with every single lot of iPSC and ESC derived lines. In addition to confirming the sterility and viability of every lot of stem cells, ArunA products are validated with FISH, DNA fingerprinting and karyotyping analyses. FISH analysis is a sensitive screening tool that detects early changes in the cytogenetics of a cell line before numerical or structural chromosomal aberrations can be revealed by karyotyping^[2]. These tests are performed on every lot of ArunA's cell lines to ensure that they reach the end user with a normal karyotype, assurances of its parental lineage and lack of culture cross-contamination, and viral or mycoplasma contamination.

ArunA's exceptional standards of quality control ensure that only cell products of the highest quality and biological relevance are provided to investigators. As examples, our hNP1™ neural progenitor cells are >90% nestin positive and <5% Oct-4 positive. They are also karyotypically stable out to ten passages and routinely tested for their ability to differentiate into bIII-tubulin positive neurons. The hMPro™ mesenchymal progenitor cells are >80% CD90, CD105, CD166 and CD73 positive and <5% Oct-4 positive and have demonstrated that they are capable of differentiating into chondrogenic and osteogenic cell types.

Stem cells have distinctive qualities that offer unique glimpses into physiologically relevant events. Unlike immortal cell lines that can harbor genetic and other abnormalities, stem cell progenitors that pass stringent quality control measures like those applied at ArunA are free of detectable aberrations. Immortal cell lines also often fail to terminally differentiate. For instance, PC12 cells will differentiate into cells with neuronal characteristics when exposed to Nerve Growth Factor (NGF), but in some cases these cells lose their neuronal phenotype when NGF is withdrawn and re-enter the cell cycle. This departure from the targeted cell type behavior weakens the conclusions that can be drawn from studies that rely on this type of neuronal cell culture.

Primary cells in culture have the advantage of retaining a stronger correlation with in vivo environments, but for HCS/HTS applications, this approach is too labor intensive and saddled with the need for many animals. Stem cell research combines both the scalable and renewable properties of stem cells and the most accurate presentation of in vivo cell biology that in vitro tools can offer. The potential of these cells to direct drug discovery toward viable therapies and insights is too valuable to lose any revolutionary discoveries to the use of substandard cell lines. The exhaustive testing of stem cell products offered by ArunA reflects our commitment to helping researchers make every experiment successful, whether the news is good or bad.

1. J.S. Draper et al, "Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells" (2004). Nat Biotech. 22(1):53-4

2. L.F. Meisner, J.A. Johnson, "Protocols for cytogenetic studies of human embryonic stem cells" (2008). Methods 45: 133-141.

The Role of Culture Conditions in Human Mesenchymal Stem Cell Metabolism during Expansion and Osteogenic and Chondrogenic Differentiation

2011-02-03T16:15:00.004-05:00

By: Jamie Chilton, PhD

Multipotent human mesenchymal stem cells (MSCs), distinguished by their ability to differentiate along multiple pathways including osteogenic, chondrogenic and adipogenic lineages, have been identified as promising candidates for advancing drug discovery and cellular therapy strategies. Most commercially available sources of human MSC are derived from bone marrow, where MSCs reside under hypoxic physiologic conditions (4-7% oxygen). Yet, MSCs are typically cultured in vitro under normoxic conditions (20% oxygen), leading to senescence and reduced population doublings compared to cells under hypoxia (1-5). Thus, a recent study conducted by Pattappa and colleagues (6) explored the effects of culture conditions on the expansion capacity and differentiation potential of nonclonal, heterogenous populations of human bone marrow derived MSCs. Oxygen consumption, glucose consumption and lactate production were all measured during in vitro hMSC expansion and differentiation into osteogenic and chondrogenic lineages under normoxic conditions. Pattappa et al found that during expansion, MSC significantly used oxidative phosphorylation in addition to glycolysis, as indicated by a 75% azide-sensitive oxygen consumption rate of ~98 fmol/cell/h. MSCs subsequently differentiated toward an osteogenic lineage for 21 days revealed no significant changes in oxygen consumption, retaining a rate of ~98 fmol/cell/h. In contrast, MSCs shifted to predominantly glycolytic metabolism and significantly lowered their oxygen consumption rate to ~12 fmol/cell/h following 21 days of chondrogenic differentiation in a more nutrient-restricted 3D pellet configuration. Osteogenic and chondrogenic cultures displayed the typical positive staining for alkaline phosphatase activity, calcium deposition and GAG deposition. Pattappa et al concluded that differentiated MSC cultures under normoxic conditions appeared to balance oxidative phosphorylation and glycolysis, and that culture conditions, not differentiation, may explain differences in energy metabolism between chondrogenic and osteogenic cultures.

In light of this recent study, ArunA’s human embryonic stem cell derived hMPro™ Human Mesenchymal Progenitor Cells demonstrate a unique property under in vitro normoxic culture conditions in comparison to other commercial sources of human bone marrow-derived mesenchymal stem cells (BM-MSC). After an initial 2 week expansion period in standard mesenchymal stem cell culture conditions, early results show that hMPro™ cells display a significantly higher change in cell density over a 96 hour time period as compared to BM-MSC. The enhanced proliferative capacity of hMPro™ cells allows researchers to meet their specific expansion needs for their projects which is difficult to achieve with other sources of MSC. Interestingly, this enhanced proliferative capacity does not interfere with the ability of hMPro™ cells to differentiate along osteogenic and chondrogenic lineages, as demonstrated by positive von Kossa and Alizarin Red S staining of calcium deposits and positive Alcian Blue staining of GAG deposition, respectively. Consequently, ArunA’s hMPro™ cells will provide an additional effective in vitro model for further studies to elucidate mesenchymal metabolism and differentiation, as well as optimizing strategies for orthopedic tissue engineering and regenerative medicine applications.

Stay posted for more upcoming information on the unique properties of our hMPro™ Human Mesenchymal Progenitor Cells.

(1) Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T (2006). Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. Journal of Cellular Physiology 207:331-339.

(2) Grayson WL, Zhao F, Bunnell B, Ma T (2007). Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun 358:948-953.

(3) Lennon DP, Edmison JM, Caplan AI (2001). Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. Journal of cellular physiology 187:345-355.

(4) Moussavi-Harami F, Duwayri Y, Martin JA, Moussavi-Harami F, Buckwalter JA (2004). Oxygen effects on senescence in chondrocytes and mesenchymal stem cells: consequences for tissue engineering. Iowa Orthopaedic Journal 24:15-20.

(5) Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral JM (2010). Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol 223:27-35.

(6) Pattappa G, Heywood HK, de Bruijn JD, Lee DA (2010). The metabolism of human mesenchymal stem cells during proliferation and differentiation. J Cell Physiol Dec 28.
http://www.ncbi.nlm.nih.gov/pubmed/21190253

A novel HTS-compatible assay for cell migration of neural progenitors

2010-12-10T17:00:00.002-05:00

By: Allan Powe, Jr. PhD

Cell migration is an important part of many normal and pathological morphogenetic processes, including central nervous system (CNS) development. Neural progenitors are created in proliferative zones within the brain and migrate to specific destinations guided by various extracellular cues and neurotrophic factors (1, 2). In addition, some developmental neurotoxicants have been shown to interfere with the migratory behavior of neural stem cells (e.g., ref. 3). The ability to identify cell migration stimulators and inhibitors can be useful for developing novel neuroregenerative therapies, identifying neurotoxicants, and testing drug candidates for safety.

To meet that need, ArunA and the team at Platypus Technologies are developing an assay for identifying cell migration stimulators and inhibitors using ArunA’s hNP1™ human neural progenitors and Platypus’ novel HTS-compatible 96-well based cell migration assay platform (Oris™ Cell Migration Assay). Early results demonstrate that cytochalasin D, an actin polymerization inhibitor, can block hNP1™ migration. Our preliminary results also show that certain growth factors, singly and in combination, can have potent chemokinetic effects on hNP1™ migration. Hence, this combination of the Oris™ platform and ArunA’s neural stem cells will provide a powerful tool to readily identify stimulators and inhibitors of neural progenitor migration.

Even as further development of this assay continues, our progress to date will be presented in poster format (ID# 2089/B409) at the American Society for Cell Biology meeting in Philadelphia, PA on December 14th 2010.

(1) Marín O, Rubenstein JL. (2003) Cell migration in the forebrain. Annual Review of Neuroscience 26:441-83.

(2) Marín O, et al. (2003) Directional guidance of interneuron migration to the cerebral cortex relies on subcortical Slit1/2-independent repulsion and cortical attraction. Development. 130(9):1889-901.

(3) Moors M, et al. (2009) Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing. Environmental Health Perspectives 117(7):1131-8.

Human neural progenitors as model systems for neurogenesis and Wnt signaling

2010-11-10T14:20:00.008-05:00

By: Allan Powe, Jr. PhD

Neurogenesis is an important process for nervous system development and maintenance. Since this process persists in adults, pharmacological control of neurogenesis could represent an approach for the treatment of neuronal loss in neurodegenerative diseases, such as Alzheimer's. Numerous studies have highlighted the importance of Wnt signaling to neurogenesis in neural progenitor cells. In particular, one recent study by Yoshinaga and colleagues (1) looked at the effect of Wnt3a on proliferation and neuronal differentiation in mouse hippocampal neural progenitor cells. They showed that Wnt3a accelerates cell division rather than promotes differentiation per se. Increased cell division leads to more cells entering the differentiation process. This study demonstrates the potential for using neural progenitor cell lines, such as the hNP1™, for further investigation of Wnt3a signaling during neurogenesis and for the development of drug targets that regulate Wnt3a signaling. In fact, two other recent studies have shown that well established and newly developed inhibitors of GSK-3 beta, a kinase that negative regulates Wnt signaling, can increase proliferation of human neural progenitors (2,3). Together, these studies indicate the potential for using human neural progenitors, such as the hNP1™, in early drug discovery for targets affecting neurogenesis.

(1) Wnt3a promotes hippocampal neurogenesis by shortening cell cycle duration of neural progenitor cells. Yoshinaga Y, Kagawa T, Shimizu T, Inoue T, Takada S, Kuratsu J, Taga T. Cell Mol Neurobiol. 2010 Oct;30(7):1049-58. Epub 2010 Jun30
http://www.ncbi.nlm.nih.gov/pubmed/20589426

(2) Novel indolylmaleimide acts as GSK-3beta inhibitor in human neural progenitor cells. Schmöle AC, Brennführer , Karapetyan G, Jaster R, Pews-Davtyan A, Hübner R, Ortinau S, Beller M, Rolfs A, Frech MJ. Bioorg Med Chem. 2010 Sep 15;18(18):6785-95. Epub 2010 Jul25.
http://www.ncbi.nlm.nih.gov/pubmed/20708937

(3) Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells. Lange C, Mix E, Frahm J, Glass A, Müller J, Schmitt O, Schmöle AC, Klemm K, Ortinau S, Hübner R, Frech MJ, Wree A, Rolfs A. Neurosci Lett. 2010 Nov 4. [Epub ahead of print]
http://www.ncbi.nlm.nih.gov/pubmed/21056624

Intro

2010-11-05T09:54:00.000-04:00

Welcome to ArunA's new blog! In case you haven't heard, ArunA Biomedical, Inc is privately held biotechnology company located in Athens, GA. We provide tools and services for the stem cell research community. If you'd like to know more about us and the products we offer, you can visit our website, or follow us on Facebook and Twitter.
We are always open to comments and suggestions about how we can serve the research community better. Feel free to e-mail us at info[at]arunabiomedical[dot]com.