On a LinkedIn discussion board I recently explained the difference between CFUs and stem cell counts and a medical device rep pinged me to ask if I would explain more, as he found that explanation helpful. One of the more confusing things out there these days are references to stem cell numbers. 99% of the docs using stem cells have no idea what these numbers mean, so neither do patients. If you were lost at the term “CFU”, you’re not alone! Your doctor likely has no idea what it means either.
Some Basic Background to Understand Stem Cell Numbers and Counts
Stem cells as they are harvested from any human tissue are usually pretty rare cells. The two most common counts you will see are from bone marrow and fat, so this is where I’ll focus. After the cells are harvested from either source, something is done to get them into a concentrated state where they can be harvested. For fat this usually means chemical digestion and then centrifugation for bone marrow it’s just centrifugation. An extra digestion step is needed to liberate stem cells from fat (the cells are locked in the matrix of collagen that gives the fat structure). For bone marrow, the cells come out in a liquid form, so this digestion step isn’t needed. In fact, it’s this digestion step that makes stem cells from fat a federally regulated prescription drug that must first have FDA approval before use or your doctor is using these cells in violation of federal drug laws (which is commonly done these days). Centrifugation means just spinning cells in a centrifuge and using gravity to concentrate them.
For either fat or bone marrow, what you end up with is not a pure population of stem cells, but a mix of cells where the stem cells are rare among many other cells types. For fat this mix of cells is called SVF (Stromal Vascular Fraction) and for bone marrow it’s usually called BMC or BMAC (Bone Marrow [Aspirate] Concentrate). Neither of these are made up of predominantly stem cells, although both are stem cell enhanced and concentrated over the stem cell number that would normally be found in their native unaltered tissues (fat and bone marrow). In particular for fat, many physicians have taken to skipping the digestion step because of the regulatory issues, but this doesn’t give viable stem cells. It’s just gives you a simple fat graft.
Actually Counting Stem Cells
Counting the stem cells in these mixes in very confusing for doctor and patients alike. Since 99% of doctors who use stem cells don’t have an advanced lab where these counts can be performed, they have to rely on what they have heard. This creates a game of physician telephone where somebody who may have known what all of this means tells another doctor who tells another doctor and so on… Suffice it to say that the guy on the end gets a garbled message.
First, you can count the number of total cells in these mixes. These are usually referred to as “Nucleated Cell Counts”. They have high numbers in the hundreds of million to billions, but they’re only a rough representation of the total number of stem cells in the mix and not stem cells counts per se. Many doctors and patients believe these are stem cell counts-they are not.
The oldest way to count the actual stem cells in the mix (which dates back to the original bone marrow stem cell research in the 1960s) is CFUs or Colony Forming Units. What the heck is that? The kind of stem cells we’re discussing here (mesenchymal stem cells or MSCs) are adherent cells-which means they love to attach to surfaces, which is how they repair tissue. In culture, these stem cells are selected out from the other cells by their ability to adhere to a plastic flask. You literally place the mix in an incubator and wait 3-7 days for the MSCs to attach to the flat flask (called a monolayer culture flask) and then discard the rest of the non-adherent cells by literally pouring them out of the flask. The MSCs that attached to the flask will form colonies (basically little collections of cells that group together), hence the term CFUs. You can, at that point, simply count the number of colonies that have formed and this gives you a number. The more CFUs you have, the more the stem cells there were in the original mix. It’s a pretty rough and dirty metric that’s more qualitative than quantitative, i.e. flask A had more stem cells which went on to form more colonies than flask B. If all your lab has is basic capabilities, you’re done at that point. If you want a more accurate count, you actually have to try and count the number of cells that attached and grew, but this is harder.
To count the number of actual stem cells and not just the groups of cells (Colonies), you usually use an enzyme to get them off the surface of the plastic and collect them via a centrifuge (a P0 cell count). You can then re-suspend them and place them on a device for counting cells. This is a whole lot more accurate and yet still a bit rough. Let me explain. Say you counted two CFUs, but one had 24 cells and the other had 12. However, another flask may have had 84 cells in one colony and 12 in another-which is also a CFU of two. However our second flask actually has a bunch more cells. However, also realize that the purpose of a CFU count was to try and get a rough metric of the number of adherent stem cells in the original mix, with the assumption that all of the MSCs attached to the plastic flask and formed colonies, so here we can say that these two samples we tested had close to the same number of MSCs. However, what those MSCs did was quite different-the cells in one flask grew much better than the other. In summary, here you begin to see the problems in these counts. CFU counting tends to have a lot of wiggle room for error-i.e. did all the cells attach to form colonies? So does counting all the cells in the colonies-some cells may have grown faster than others.
Another way to count cells is with flow cytometry. This is a very fancy machine (we have one) that uses markers on the stem cells to try to identify them. The cells are first exposed to a fluorescent dye that binds to a specific marker on the cell that’s commonly found in stem cells. You then run the marked cells through a channel (one by one, but crazy fast) and the machine counts how many of the cells have a certain marker using LASER light. However, since there is no hard and fast single cell marker that’s only found on MSCs, all you can get a rough quantification of the number of cells highly likely to be MSCs by using four common markers. Regrettably some of these markers are found on other cells, so the count is not 100% accurate.
Finally, you can also use FACS (Florescence Activated Cell Sorting-we also have one of these) to sort individual labelled cells. This is a similar technology to flow cytometry, however, this machine can sort cells with certain markers into separate containers. You can then use flow cytometry or more sorting to get a very pure population of cells to count. Hence FACS is likely the most accurate way to count cells.
In summary, note that most companies that claim to have counted the number of stem cells in the mix of cells that you get from fat or bone marrow have only performed the much less accurate CFU counts because they’re cheaper and easier. Some do flow cytometry counts, but as I’ll explain later for fat stem cells, this is fraught with issues. Few take the time to actually count the cells in the colonies or use FACS.
Is What You Just Counted Alive or Dead?
So you just tried to count CFUs, the actual stem cell numbers in the colonies, and you even counted the cells in the SVF or BMC with a flow cytometer. Feeling pretty good that you have a very accurate stem cell count that actually will translate into something in the clinic? Not so fast!
The next question is whether the cells are alive or dead. It wouldn’t be good if you thought you had 1 million cells only to find that half were DOA (Dead on Arrival). So the next step is a viability count. This is usually a simple stain that’s taken up by dead cells while living cells can pump it out. This is known as “viability testing” using live/dead counts. Your cells should be at least 85%-95% viable.
However, this doesn’t capture another important metric. This one I can demonstrate by using patient examples to represent the cells. Let’s say we have three cells, one represented by a patient who is healthy, one by a patient in the ICU, and the second by a patient who is dead. Our viability counts says that we have two good cells (i.e. both are technically alive), but a more sophisticated real world analysis shows us that we have only one cell we would want to use, as the sick ICU cell isn’t going to do much. So we also need to know which cells are healthy. Regrettably, even research papers performed in universities screw this one up. This is exemplified by a recent research paper that showed that certain anesthetics were OK with stem cells, but the researchers only looked at live/dead counts instead of the more sophisticated healthy, sick and dying, and dead counts (live/apoptotic/dead). When our advanced research lab redid the anesthetic stem cell experiment with the more sophisticated testing, the results came out differently. Getting this healthy count requires more sophisticated staining and testing.
Another way to look at the health of the stem cells is to put them back in culture and continue to grow them. When you have the cells at the CFU stage, that’s called P0. If you count the cells one by one at that stage, it’s often called a P0 count. The “P” means “Passage” which is a short hand notation for when the cells are fed (usually every 2-4 days). If you grow them out for another 6 days and feed them twice more in that time, you would be at P2 (Passage 2). This is yet another way to make sure you have live cells and compare the cell content of one cell source over another. However, realize that since as discussed above, some types of stem cells grow faster than others, this can issues with comparing these counts.
In summary, counting stem cells isn’t enough, you also need to know if the cells are alive or dead. In addition, add to that healthy, dying, or dead. However, there’s a whole other metric-differentiation assays. Wow, this is getting complex!
Measuring what the stem cells in the mix can do is really advanced work, so if clinics rarely get as far as counting the number of nucleated cells in their samples or even counting CFUs and almost none own a flow cytometer, you can bet they never ventured to figure out if the stem cells in the mix work as advertised. One of the major functions of stem cells is that they can turn into (a.k.a. differentiate into) other cell types. If our focus for this discussion is arthritis, then the stem cells in the mix had better be good at differentiating into at least cartilage and bone. However, this is where fat stem cells often fail-as they’re horrible at turning into these orthopedic tissues when to compared to the much more facile bone marrow. While fat stem cells can do this, they need a very heavy handed culture stimulus to accomplish the feat, akin to a sledgehammer of artificial stimulants when compared to the gentle nudge that bone marrow stem cells need. Almost none of the fat stem cell clinics out there are capable of providing this stimulus in culture.
In addition, if you don’t want to sit around and wait for cells to become cartilage, bone, or fibrous tissue cells and then form cartilage, bone, or fibrous tissue, you can look at gene expression. This requires a quantitative PCR machine (we have one) that looks at the genes that are expressed in the cell. These strands of RNA produce the proteins that make the cartilage, bone, or fibrous tissue components. So rather than waiting months for these things to form, you can also check to see if the genes that produce these things are being used by the cell.
So in summary any sophisticated stem cell count of a mixture of cells in adipose same day SVF or BMC needs:
1. The total nucleated cell count-of which a small fraction of the total cells are stem cells (the actual stem cell number is unknowable from such a count). Very few clinics make any attempt to count these total cells.
2. CFUs are helpful, but only as a qualitative rough metric, not as an exact count of anything. They tell you roughly how many stem cells were in the sample and are better for comparing the counts in mix A vs. B.
3. Stem cell counts out of colony formation (commonly referred to as P0 counts) are more accurate and are simply a way to count the number of cells in those colonies. However, few companies, labs, or clinics either take the time, spend the money, or know how to accomplish this next step.
4. Flow cytometry counts of cells that have stem cell markers are another good metric to confirm cell numbers. However, since there is no one common MSC marker, you get a quantitative count that gives you a good idea of stem cell numbers, but not an exact count as some non-stem cells can be counted.
5. Growing cells out further in culture can give you information which also takes into account cell health (i.e. dead cells don’t grow).
6. Live/dead viability counts are a quick and dirty way of determining how many of the cells you’ve counted are even alive.
7. More sophisticated live/dying/dead counts are an even better way to look at stem cell viability as they focus on healthy cells.
8. Differentiation assays or gene expression are good ways to look at whether the healthy cells can likely repair what you want them to repair.
Fat versus Bone Marrow Stem Cell Counts: Now the Confusion Really Begins!
As you can see from above, this issue is a lot more complex than it looks. In addition, it’s more confusing for doctors and patients alike when the proponents of fat stem cells claim that fat has more stem cells hence it must be better at everything. There are a few issues with that argument given what you learned above and adding in the following data:
1. Fat stem cell counts are usually wildly inflated by liposomes/micelles. These tiny bits of oil are often counted as cells when they are really just junk. There’s a nice paper on this issue here.
From that paper: “Intrigued by the high cell numbers (5 to 20 million cells/gram) reported by kit/device manufacturers such as MediVet-America (Lexington, KY), Intellicell Biosciences (New York, NY), and Adistem, Ltd. (Hong Kong) in adipose stem cell therapy compared to other methods (e.g., Chung,Vidal, and Yoshimura), INCELL staff conducted a research study to investigate the high apparent yield of stem cells. “ and…
“This study shows that incorrect counting of adipose derived SVF cells and the subset of regenerative stem cells can subsequently result in inaccurate dosing…The nature of adipose tissue itself with variability in dissociation by enzymatic digestion can all contribute to the outcomes. Fat tissue has a propensity to form acellular micelles and oils upon tissue disruption. Processing methods or reagents (e.g., Solution E or lecithins) can generate micelles that may be erroneously counted as cells.”
2. Fat stem cells are very hard hit by metabolic syndromes and are often harvested the wrong way. Basically, if you’re fat and want liposuction, your fat stem cells aren’t as active and able to repair tissue as your skinnier counterparts. In addition, typical liposuction can hammer stem cells.
3. The anesthetics commonly used in liposuction are very toxic to stem cells. So they numbed you up to make you comfortable, but did the numbing agent kill many of the stem cells?
4. There are a lot more stem cells in bone marrow than anybody ever thought. First there are two main stem cells types-MSCs and HSCs-both valuable for orthopedic purposes. In addition, there’s also a small stem cell variety. Even when it comes to MSCs, a recent paper shows that our counting methods using flow cytometry likely have dramatically under counted the MSCs in bone marrow.
5. There’s a second population of MSCs in bone marrow that only the Regenexx-SD procedure isolates, increasing our total MSC counts to 5-10 times higher than techniques that only use the traditional buffy coat isolation method (all bedside centrifuges and techniques used by other clinics).
The issue of how many stem cells are in a sample of tissue is very confusing to patients. In addition, few physicians understand the subject in any depth. I hope this quick review of the subject helps everyone get on the same page!