Frequently Asked Questions
- Aside from Morphology and Microscopy how are the MLPC clonal cell lines characterized?
- Can you detail some of the key differences between MLPCs and the MSC stem cells?
- Do you know the functional difference of the MLPC leukocyte phase and MLPC fibroblast phase? Do the MLPCs lose cell functionality when they go to the fibroblast phase? Additionally, there is loss of expression of the SC markers during this phase. Any ideas
- Are there any marker combinations unique to the MLPC. What makes these cells so unique?
- What pathogens have the MLPC's been tested for?
- Have you seen chromosomal rearrangement after they have been cultured for a while?
- What are your quality controls measures? What kinds of QC/testing are performed to make sure only quality cells are delivered every time?
- What kind of clonality do they have?
- Are the MLPCs cloned during the leukocyte or fibroblast phase?
- Is the medium provided by BioE to keep the stem cells undifferentiated?
- Can you divulge specific amounts of medium used to express the MLPC's down the differentiation paths now or is this information only provided with a sale of MLPC's?
- What kind of contact have the MLPCs had with animal cells in culture?
- What happens with confluency? Do they only slow down? Do they come back if you split them into trays after confluency?
- Can MLPC's be raised in suspension?
- Have you differentiated MLPC into epithelial cells? If not, are you planning to and when?
- Have you differentiated MLPC into cartilage cells? If not, are you planning to and when?
- Is anyone working on differentiating MLPCs into cardiocytes?
- Can MLPC's be viral infected?
- Has anyone done any in vivo work to date?
To date, we have characterized the MLPC clonal cell lines by following morphology throughout culturing, through CD surface marker staining throughout culturing, and through gene microarray analyses. Once MLPCs have been differentiated into a specific cell lineage, we perform microscopy to stain either for surface markers (where applicable) or intracellularly (where applicable). We have not performed any gene microarray analyses on the differentiated cells.
Early isolated MLPCs are CD34, CD45, CD9, SSEA-3, SSEA-4, STRO-1, SCF, and CD133 positive. MSCs are negative for these. MLPCs can be differentiated into all three embryonic layers. MSCs are limited to mesodermal differentiations, although some researchers have claimed further differentiation capabilities. The MSCs require culturing densities higher than MLPCs, and are not capable of as many doublings. In addition, there's a differential expression of 360 genes as determined by gene microarray analyses.
Do you know the functional difference of the MLPC leukocyte phase and MLPC fibroblast phase? Do the MLPCs lose cell functionality when they go to the fibroblast phase? Additionally, there is loss of expression of the SC markers during this phase. Any ideas
We do not know the functional difference between the early leukocyte phase and the fibroblast phase. There are too few cells to perform differentiations on them at the leukocyte phase. It is during the fibroblast phase that we initially perform differentiations to determine their capabilities. From there, we proceed to cloning and perform differentiations throughout various passages to ensure that they are still retaining their differentiation capabilities. Yes, we lose the CD34, CD45, SSEA-3, SSEA-4, STRO-1, SCF, CD133 in the fibroblast phase, but again this is when we perform our initial differentiations, so no they don't seem to lose their multi-potency. In addition, our gene microarray analyses indicate that the MLPC clones retain their stemness.
The MLPCs early in culture are CD34, CD45, SSEA-3, SSEA-4, STRO-1, SCF, CD9, and CD133 positive. Because they are CD34 and CD45 positive early on and their growth and morphology differ from other cells previously identified by other groups, distinguishing them we believe, as a different population. In addition, they have a normal and stable karyotype; they are capable of differentiation into all 3 embryonic layers; they have been cloned from a single cell; they grow easily in standard medias; they provide consistency; and they are commercially available.
All by qPCR testing: HIV-1 DNA, HIV-2 DNA, HBV DNA, HCV RNA, CMV DNA, and Mycoplasma DNA.
We have performed gene karyotyping on the MLPC clonal cell lines to determine genetic stability. The results have shown that the cells are genetically stable and have no deletions or mutations. This was performed on both early (passage 4) and later passaged (passage 18) clones.
What are your quality controls measures? What kinds of QC/testing are performed to make sure only quality cells are delivered every time?
We follow the cells from early culturing and throughout observing morphology and growth characteristics. We perform immunophenotype staining throughout the culture at various time points. Cells are then differentiated into the 3 germ layers. Once differentiation is confirmed, they are cloned. Cloned cells are then differentiated into the representative 3 germ layers; phenotyping is performed as well. These tests are performed on early and late passaged cells for confirmation. In addition, gene expression and genetic stability by microarray are performed. Cells are also tested for HIV-1 DNA, HIV-2 DNA, HBV DNA, HCV-RNA, CMV-DNA, mycoplasma DNA and sterility.
Our MLPC clones were derived from a single cell culture using standard limiting dilution methods of 1 cell/well, 30 cells/96 well microtiter plate.
During the fibroblast morphology stage.
BioE will not be providing any medias at this time for growing the MLPC's or for differentiating them. We do, however, provide all the information that pertains to the medias and their manufacturers, growth factors, etc. that we have used to successfully grow and differentiate the MLPC's. MLPCs do not require special medium in order to prevent differentiation; they do not spontaneously differentiate without specific tissue-specific stimulus.
Can you divulge specific amounts of medium used to express the MLPC's down the differentiation paths now or is this information only provided with a sale of MLPC's?
Yes, all information on differentiation medias is included with the Methods Book that is shipped with the MLPCs to customers. It will also be available to anyone on the BioE website.
We do not culture the MLPCs with any other cell types, human or animal. They are derived from human post partum umbilical cord blood and are cultured with medias only. They have however been grown in medium containing fetal bovine serum.
What happens with confluency? Do they only slow down? Do they come back if you split them into trays after confluency?
At confluency the MLPCs will slow down in their growth rate. Yes, if they are split they resume their typical growth rate. Repeated growth to confluency may permanently affect their growth and ability to differentiate.
In standard growth medium used the cells do not grow in suspension.
No, we haven't performed epithelial differentiations. We will potentially perform these differentiations, however, they are not ones that we are currently working on. For now, we're making the MLPCs available for groups to work on both the differentiations that we have and those that we haven't done.
No, we haven't performed cartilage differentiation internally at BioE, however, one of our collaborators has differentiated them into chondrocytes (cartilage). Internally, we have performed bone (osteogenic),fat (adipogenic), and skeletal muscle differentations, all of which are in the mesodermal layer as is cartilage.
Our endothelial differentiation is specific for microvasculative and coronary artery endothelial cells. We do plan to look at cardiomyocytes soon.
Yes, we have a collaborator who has performed transfections with telomerase reverse transcriptase and with GFP.