Tuesday, April 19, 2011

Growing Tall by dedifferentiating cells into Pluripotent Stem Cells

Many have inquired that whether it's possible to induce height growth via bone modeling.  The bone completely replaces itself every ten years and bone is living tissue then shouldn't it be possible to completely replace the bone with a taller and longer bone?  The issue is that bone is primarily made up of Type I Collagen which is more matrix than bone.  That tissue it would be possible to grow taller via gap repair.  Remember that Marfan's Syndrome which results in tall stature involves a mutation involving genes related to Type I Collagen so there's anecdotal evidence of Type I collagen being able to increase height.   It's the osteoblasts and osteocytes that secrete the Type I Collagen bone matrix so it's those cells that ultimately have to be the ones that need to be reprogrammed in order to make a taller bone.

One way to do that is to cause the dedifferentiation of osteoblasts and osteocytes into Pluripotent Stem Cells and then get them to differentiate into chondrocytes.  Which would then secrete Type II collagen and a cartilagenous template could be formed much like the initial bone begins entirely by cartilagenous template.  If the Type I Collagen matrix proves to be an obstacle then it can be demineralized with acid.  This cartilagenous template would be much bigger than when you were a baby and you would grow much taller from this template as the template would be much bigger than when you were a baby.

But since bone is constantly being remodeled then the type I collagen would gradually be removed by the osteoclasts and the chondrocytes in place of the osteocytes would now be secreting Type II collagen and the bony template would gradually become a cartilagenous one and you would grow taller in your bone like you were a baby. The problem is that osteoblasts and osteocytes secrete TGF-Beta1 which is important for chondrogenesis.  TGF-Beta1 results in Smad 2/3 Phosphorylation.  So with no osteoblasts and osteocytes TGF-Beta1 levels would be lower discouraging chondrogenesis.  However, it's possible to use external sources of TGF-Beta1 and chondrogenesis can occur without TGF-Beta1 just not as well.

So how do we induce dedifferentiation of osteoblasts and osteocytes(we are of course using LSJL as an inducer of chondrogenesis)?

Influence of low oxygen tensions on expression of pluripotency genes in stem cells.

"[Stem cells] are residuing in defined microenvironments - "stem cell niches". The embryonic stem cells (ESC) are derived from embryos which exist in 3-5 percent oxygen condition. This environment is physiologically normal not only for ES cells but also for many other types of stem cells. These observations suggest that low oxygen condition plays a very important role in the maintenance of cell stemness. Pluripotency is regulated by the family of hypoxia inducible factors (HIFs), which are dependent on oxygen tensions[The initial primary ossification center is an hypoxic environment and the growth plate is also hypoxic]. HIF-2-alpha is an upstream regulator of Oct4, which is one of the main transcription factors used to generate the first induced pluripotent stem cells (iPSCs)[So HIF2-alpha upregulates Oct4 which can induce stem cell pluripotency]. It has been shown that knock-down of HIF-2-alpha but not HIF-1-alpha, leads to a decrease in the expression of Oct4, Nanog and Sox2[HIF1-alpha is important for inducing chondrogenesis whereas HIF2-alpha is important for inducing dedifferentiation of stem cells], which are important stem cells markers. The structure of hypoxia inducible factors as well as their behavior in hypoxia and normoxia was described. Therefore optimization of oxygen concentration seems to be crucial from the stem cell transplantation as well as iPS transplantation standpoint. Although many experiments with cell culture under low oxygen condition were performed, there is still much that is unknown. This short review presents some aspects on important issue of hypoxia induced regulation of stemness."

"cells grown in higher  oxygen tension (20% O2) accumulated more mutations  than cells in culture at 3% O2. Most of the mutations were transversions (G:C to T:A), which is a marker of mutation of oxidative DNA damage"

So inducing expression of HIF2-alpha can induce cellular dedifferentiation.  Remember, it's PHD that degrades the Hypoxia inducible factors in high oxygen environments.  So a PHD inhibitor is more important than the oxygen content.  Elevated levels of oxygen just enable PHD to work.  It's also possible that if you increase the levels of the Hypoxia inducible factors enough then they will be able to perform their actions properly regardless of being degraded by PHD.

Let's look specifically at osteoblast dedifferentiation.

Runx2, p53, and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre-chemotherapeutic osteosarcoma cell line (OS1).

"Osteosarcomas are the most prevalent primary bone tumors found in pediatric[children] patients. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression[Parathyroid Hormone targets Zfp521 which inhibits Runx2 activity, Runx2 encourages osteoblast differentiation, PTH, Zfp521, and p53 overexpression may be ways to cause osteoblast dedifferentiation], while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS-2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development."

 p53 and pRB seem essential for proper cell growth and preventing cancerous tumors if you increase their expression you will potentially lower Runx2 expression, cause osteoblast dedifferentiation, and prevent osteosarcoma.

"the elevated levels of Runx2 present in OS1 cells may promote blood vessel growth in osteosarcoma cells to support tumor growth and increase its metastatic potential"<-so Runx2 increases blood vessal growth which may increase the oxygen content discouraging height growth and VEGF-A seems essential to height growth.  HIF2-alpha upregulates VEGFA.  Knockout of VEGF-A causes cell death in chondrocytes.  You might need permanently elevated levels of HIF1-alpha and HIF2-alpha as loss of VEGF-A may cause chondrocyte cell death.  The blood vessal growth induced by Runx2 may inhibit that.

Shear stress also induces Runx2.  LSJL induces fluid shear stress.  So LSJL itself may inhibit the dedifferentiation of osteoblasts meaning that an alternative method of inducing chondrogenesis may be needed if attempting to dedifferentiate osteoblasts.

Growth and cellular differentiation: a physico-biochemical conundrum? The example of the hand.

"Currently, the predominant hypothesis explains cellular differentiation as an essentially genetic intracellular process[this is the process we are operating on, we are trying to activate genes in stem cells to induce chondrogenesis with LSJL]. The goal of this paper is to suggest that cell growth and differentiation may be, simply, the result of physical and chemical constraints. Bone growth occurs at the level of cartilage conjunction (growth plate) in a zone of lesser constrain[So changing the level of constrain is a mechanism of growing taller, distraction osteogenesis changes the level of constrain, however this hypothesis doesn't explain growth abnormalities like gigantism]. It appears that this growth also induces muscle, tendon, nerve and skin elongation. This cartilage growth by itself seems to explain the elongation of the hand. Growth stops at puberty likely because of feed-back from an increasing muscle load[again Gigantism and myostatin deficient organisms would seem to refute this hypothesis as they are very tall and muscular]. The ossification (that is differentiation of cartilage into bone) appears to result from the shear stress induced[this is true, however, new stems can differentiate into cartilage replacing the ossified ones, this muscle load only increases growth rate and not adult height]. The study of bone age, obtained by X-ray picture of the hand, shows that ossification of epiphyses is very precise both in time and space. Computer modelization suggests that this ossification occurs where shear stress is greatest. The cartilage which does not ossify (joint, nose, larynx, ear, bronchus, etc.) is not exposed to high shear. Shear stress induces the secretion of extracellular matrix and a change of the biochemical environment of the cell. Precipitation of calcium phosphate, as in ossification, seems related to the alkalosis induced by shear stress. To speak in more general terms, loss of cellular differentiation, as occurs with cancer, can result from a change in the physical-chemical environments."

This hypothesis is not entirely correct but the environment may play a role in osteoblast differentiation.

"The use of rigid fixation for fracture of the extremity is common place. Epiphyses plated for a year show increased bone differentiation, premature closure and growth arrest"<-so yes the bone environment does affect height growth.  So rigid fixation reduces height growth likely because the elasticity of the bone is essential to height growth.  Some stretching of the bone is likely involved when you sleep and that can't occur when fixated.  How can hypertrophic chondrocytes push the bone apart from two ends when standing?  It likely can't.

"Extracellular matrix and/or low oxygen tension differentiates stem cells into chondrocytes"

The loss of shear stress may be enough to induce dedifferentiation of osteoblasts due to lower Runx2 levels.  As the study says, that the areas without ossification are those with lower shear stress.  So if you temporarily lowered shear stress(by say immobilization-like space flight) then would osteoblasts begin to dedifferentiate.  Then you could reinduce mobilization and grow taller than before?  However, astronauts do not experience permanent height growth.  Maybe they don't cycle enough to notice a difference.  Like say each space trip followed by landing induces a few millimeters of height growth that would not be enough to notice.


There are factors that can in general induce dedifferentiation like Oct4 and factors specific to osteoblasts like Runx2.  It may be possible to manipulate shear stress to grow taller but it wouldn't be as simple as cycling off and on LSJL.  You would have to be completely immobilized or in a low gravity environment.  It may also be possible to experiment with Runx2 inhibition or increase activity of Parathyroid Hormone or of Zfp521.


Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin.

"While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair."

Maybe we can dedifferentiate our osteoblasts into stem cells and then into chondrocytes.

"In salamanders and fish, appendages (limbs, fins, and tails) regenerate via formation of a blastema, a mass of proliferative undifferentiated cells that contains the progenitors of the regenerating tissues"

"stump osteoblasts downregulate the expression of mature and intermediate differentiation markers in response to fin amputation. Amputation in the middle of a ray segment resulted in osteocalcin:GFP downregulation and distal shift of GFP signal also in the adjacent uninjured segment, suggesting that putative signals regulating osteoblast dedifferentiation spread from the injured segment to neighboring bone segments."

" glycoprotein secreted from immature bone cells, Tenascin , was induced in osteoblasts close to the amputation plane"

4 comments:

  1. "Growth stops at puberty likely because of feed-back from an increasing muscle load[again Gigantism and myostatin deficient organisms would seem to refute this hypothesis as they are very tall and muscular]"

    So weightlifting stunts height growth at puberty?

    ReplyDelete
  2. what do you think of swimming as a gravity reduced enviornment

    ReplyDelete
  3. Annonymous #1: The study is inaccurate. The study gives a cellular mechanism for growth namely shear induced changes in genetic expression. Factors that increase shear strain like weight lifting make you grow faster but they don't stunt growth. In the chick loading study, the chicks were load continously so there growth plates weren't properly decompressing. The chicks were loaded continuously and not intermittently.

    Annoymous #2: You need to be zero gravity for a long enough time to lower Runx2 expression and still not sure if this will work. It's an interesting idea, will have to look into astronaut studies.

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  4. Hello,

    This is the perfect blog for anyone who wants to know about this topic. Pluripotent stem cells have the potential to differentiate into specialized cells in the body. These cells describe all of the cells that can give rise to the different cells found in tissues...

    ReplyDelete