This post is more of an extension of the first article “New Proposed Height Increase Method Using Gene Therapy On Engineered Pluripotent Mesenchymal Cells (Breakthrough!)“. It gives the method a more solid theoretical foundation and validity. In the first study, the researchers used recombinant adenoviral vectors carrying the human BMP-2 gene (rh-BMP2) to transduce MSCs to have a prolonged effect of increasing the expression of the protein which the gene is supposed to make.
We saw that bone formation and growth can be regulated or increased from increasing the expression of certain types of growth factors by using an implanted vector.
Update 2/16/2013: At this point I would have to say that idea I had propose in the previous post above has some theoretical backing behind it but is also quite flawed once I go back and read the studies and see what they are actually implying. Those studies showed that the growth factors being made by the mesenchymal stem cells, caused osteogenic effects, not chondrogenic. This just goes to show how science and research and better understand causes out previous understanding to be proven wrong and for us to constantly be changing our level of understanding of the science.
Analysis & Interpretation
Let’s first look at the first study. We are learning a lot from this study. It seems that the BMP-2 which we are so familiar with seem to cause bone cells like osteocytes and osteoblast than say cartilage cells if you inject the growth factor straight into say the epiphysis of long bones in a lab rabbit. The bone formation can be seen in vitro but also in vivo however for the BMP-2 to work in vivo (in the actual lab animal) it has to have really high concentrations, around the mg level of concentration instead of the micrograms which most growth factors are found in. Plus, the effects of the growth factor is really fast and short in time duration. There is an initial injection of the growth factor but that will only get diffused and degenerated after say a few days. To get around the issue of only a small dose and short time frame of dosage, the idea was to inject cells into the rabbit leg bone defects than growth factors.
The cells themselves are the Mesenchymal Stem Cells (MSCs) which can be found from a bone marrow removal and then centrifuged or have some process done to them which would cause the MSCs to be separated, filtered, and then purified. These marrow derived MSCs would be place in a culture. The vectors known as recombinaant adenoviral vector will have the human BMP-2 gene (Adv-BMP2) inside oft them already. The vectors are added into the culture. The cell membrane of the MSCs which are pluripotent area willing to let the vectors get hooked to a receptor of the outer cell membrane and either let the entire vector inside or just let the gene which the vector was housing to be popped inside into the intercellular matrix of the cell. I would guess then that gene finds it’s way to the surface of the nucleus membrane, get through that, and then somehow cause the genome and chromosomes of the nucleus to start turning on or turning off the specific genes for the specified growth factor. For this case, it is the BMP-2. The cells start expressing the BMP-2 and they start turning into aka differentiating into the bone cell type aka osteoprogenitor cells. The researchers can confirm that the MSCs are turning into bone cells like osteocytes/osteoblasts by using assays and histology to check the level of alkaline phosphatase expression, the amount of Collagen Type I, another compound called osteopontin, osteocalcin, and matrix mineralization.
At the microscopic level with say one cell or a culture of same type pluripotent MSCs this entire changing of cell type is possible using elementary gene therapy techniques.
Now let’s look at the 2nd study. Note that the 1st and 2nd studies/articles are written by the same group of university researchers so the two articles are essentially the same. Right off the bat the researchers state this point, “Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line”. This was proved from the 1st study. MSCs you find from the bone marrow can indeed differentiate into the bone cell type. They conclude by saying, “We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted.” In both articles the researchers put the cells with the vectors for expression BMP-2 into the rabbit’s intervertebral region, between the lumbar vertebrate #5 and #6.
Now let’s look at the 3rd study. Again the researchers are trying to prove that the MSCs you get from bone marrow can turn themselves aka differentiate into the bone type of cells. The setup is however a little different.
- First, there is a culture but there is two types of cultures the MSCs will be in to test for differentiation capacity and extent, a monolayer and then another three-dimensional alginate culture systems
- Second, the MSCs will be tested with getting the adenoviral vectors having both BMP-2 encoding genes and BMP-6 encoding genes.
The results show that the BMP-2 encoded genes seem to work better in getting the pluripotent MSCs to differentiate in both of the culture types for better and/or faster bone formation.
The Actual Method
In the previous post I had talked about the idea of using gene therapy to cause height increase. That was a two step process,
- First is the injection of pluripotent MSCs which would only express chondrogenic genes causing it to produce only chondrogenic growth factors. – This would hopefully result in all the stem cells being injected to turn into chondrocytes.
- Second is the injection of already differentiated chondrocytes with their nucleus getting the adenoviral vector for the up-regulated expression thus the production of growth factors which will cause the chondrocytes , both already inside the epiphysis and then next chondrocyte group injection, to come together and proliferate thus forming a new cartilage inside the epiphysis.
The idea for the previous post was a theory and a proof of concept on how to use the gene therapy techniques we know right now to help us increase height. The truth is that the elements to do the entire process is the basic process of tissue engineering in general. there is always these steps
- You take a sample from the person’s body which has some stem cells
- You put the sample through some type of machine to filter the stem cells
- The stem cells are put in a culture of a certain type to grow them to a certain number.
- You put the stem cells in some type of matrix or porous scaffold.
- You add growth factors to the stem cells to make it grow to proliferate
- You implant that porous scaffold into the human which they came from.
- You watch to see how well the human body accepts the scaffold filled with the stem cells.
This is tissue engineering. You already have many biomedical researchers doing this type of research for finding ways to regenerate or heal cartilage, but more specifically the articular cartilage in the major joints like the knees and hips.
This step in general is something which I have already done enough research to understand the basic steps of tissue engineering.
Now we are learning how to incorporate genetic engineering/ gene therapy with the tissue engineering to make the stem cells we put in either or both the scaffold or culture to make them do certain things like differentiate, proliferate, hypertrophy, release certain types of growth factors.
For the new and better improve method for height increase is to focus only on the step where the cells themselves have something done to them more than just putting growth factors with them in a culture or scaffold. It is to put a vector with a certain type of protein producing gene in them. This means that the effect for growth factor production or prolfieration will be increased. The dosage will be go along for a longer amount of time meaning that the desired process is increased. Instead of just the first dosage of growth factors effecting the stem cells, the vector will cause the nucleus of the cells themselves to go through the process intrinsically and make it more permanent, at least until the cells go through apoptosis.
Calcif Tissue Int. 2001 Feb;68(2):87-94. – Cheng SL, Lou J, Wright NM, Lai CF, Avioli LV, Riew KD.
It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation both in vivo and in vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effect in vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formation in vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both non-transduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potential in vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation both in vitro and in vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).
- PMID: 11310352 [PubMed – indexed for MEDLINE]
Calcif Tissue Int. 1998 Oct;63(4):357-60. – Riew KD, Wright NM, Cheng S, Avioli LV, Lou J.
Source – Department of Orthopaedic Surgery, Washington University, One Barnes Hospital Plaza, Suite 11300 West Pavilion, St. Louis, Missouri 63110, USA.
Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line. It has been demonstrated that BMP-2 can enhance this differentiation process. In an attempt to prolong the transforming effect of BMP-2, we used an adenoviral vector carrying the human BMP-2 gene to transduce marrow-derived mesenchymal stem cells of New Zealand white rabbits. Assays on tissue culture demonstrated that these cells indeed produced the BMP-2 protein. These transduced stem cells were then autologously reimplanted into the donor rabbits. The cells were placed in the intertransverse process area of five rabbits. In one out of the five rabbits, this resulted in the production of new bone which was demonstrable on both radiographic and histologic examination. We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted.
- PMID: 9744997 [PubMed – indexed for MEDLINE]
J Orthop Res. 2006 Jun;24(6):1279-91. – Zachos TA, Shields KM, Bertone AL.
Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.
(c) 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
- PMID: 16649180 [PubMed – indexed for MEDLINE]