(Breakthrough)Direct chondroinduction by ESC implantation

Being able to induce chondrogenesis directly by stem cell implantation would be a huge breakthrough as there are stem cell sources available in breast milk for instance or umbillical cords.

Developmental-like Bone Regeneration By Human Embryonic Stem Cell-derived Mesenchymal Cells.

The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice{This is a problem in extrapolating results to humans as humans are not immunodeficient!  The human immune system may reject stem cells}. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After six weeks, x-ray, microCT and histological analysis showed that the hESC-MCs had consistently formed highly vascularized new bone that bridged the bone defect and integrated seamlessly with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without pre-implantation differentiation to osteo- or chondro-progenitors.”

It’s not quite true that the hESCs were implanted as is into the bone as the hESCs were first differentiated into MSC-like cells which requires for instance silencing or activating some genes.

“direct transplantation of undifferentiated hESCs induces uncontrollable spontaneous differentiation and teratoma formation instead of the desired healthy, functional tissue”<-a teratoma is a tumor made of ectopic tissue.

The hESCs were more epithelial cell types whereas the hESCs-MCs were more fibroblastic cell type.

The hESC-MCs have a fibroblastic morphology resembling adult hMSCs“<-So they were like adult MSCs but with some epigenetic modifications.

“Flow cytometry analysis of the hESC-MCs for markers of adult MSCs demonstrated they were positive for CD73 (99.9%), CD90 (85.4%), CD105 (100%), CD146 (99.6%), and CD166 (100%), and were negative for the hematopoietic markers CD34 and CD45. These values were nearly identical to those obtained for the control adult hMSCs, except that the hESC-MCs had lower Stro-1 expression than adult hMSCs (0.3% vs 11%).”

“The percent of cells positive for SSEA-4 was 60% for hESC-MCs vs 35.5 % for adult hMSCs. For Oct4 the percent of hESC-MC cells expressing the marker was 85.8 vs 94.1 % for adult hMSCs, for Nanog: 67.8% positive in hESC-MCs vs 63.7% in adult hMSCs, and lastly 100% of hESC-MCs were positive for Sox 2 vs 99.9% for adult hMSCs.”

Osteogenic differentiation was three times higher for adult MSCs than for ESC-MCs.  Endochondral ossification was observed in the bone defect healing for ESC-MCs but not for the adult MSCs.

“adult bone marrow-MSCs are an adult tissue resident stem cell whose normal function is small scale tissue repair to maintain homeostasis and its own self-renewal. When extracted and cultured, adult bone marrow-MSCs will have a higher tissue specific gene expression because of their developmental lineage in that tissue. However, this also potentially limits their capacity for large-scale tissue regeneration, perhaps because of inherent functionality or even limited proliferation.”

“adult hMSCs from bone marrow are capable of tissue repair, while hESC-MC are capable of induced developmental tissue generation.”

“the hESC-MC cell morphology is similar to that of adult MSCs, although adult MSCs have more elongated filopodia[slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells].”

So the breakthrough isn’t that you can grow taller by eating umbillical cords as these cells were pre-differentiated into mesenchymal cells.  The breakthrough is that the limitation on height growth after puberty is based on the characteristics on the cells themselves.  The presence or absence of the growth plate or bone mineralization may not be the limiting factor but rather the cells themselves.

That means that any height increase modality such as LSJL should be ensured to have an effect on the cells themselves to induce them to a more developmental stem cell type.  Now stimuli induced by LSJL like hydrostatic pressure, interstitial fluid flow, and dynamic compression have all been shown to induce changes in cellular gene expression.  MSCs and hESC-MCs were largely similar between pluripotency markers Oct4, SSEA4, Sox2, and Nanog.  Differences lied mainly in the expression of Stro-1 was lower in hESC-MCs than adult MSCs.  Which is odd as Stro-1 positive MSCs tend to decline with age.

The hESC-MCs were also implanted into a defect with a scaffold so it’s unclear whether these implanted cells could generate endochondral ossification on their old without a defect nor scaffold.

Here’s some stuides on how mechanical stimulation can alter the genetic expression of mesenchymal stem cells so we can see whether LSJL does in fact prime adult MSCs to be more chondrogenic.

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement.

“[We] investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. Methods: Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB{also upregulated by LSJL}. There were changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process.

Mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation.”

Ability of the MSCs to differentiate into chondrocytes was not tested.  This type of loading increased ERK1/2 phosphorylation but not Akt phosphorylation whereas LSJL increased both levels.

“the cytoskeletal organization of the cells displayed alterations, with MSCs taking a more rounded shape when loaded for 2 h, while cells appeared more flattened with a more prominent filamentous actin network when rested for 22 h.”

Comparison of genes altered to LSJL was not done but no genes altered seemed to be involved in chondrogenesis.

Intermittent traction stretch promotes the osteoblastic differentiation of bone mesenchymal stem cells by the ERK1/2-activated Cbfa1 pathway.

“We investigated the osteoblastic differentiation of bone mesenchymal stem cells (BMSCs) affected by intermittent traction stretch at different time points and explored the mechanism of osteoblastic differentiation under this special mechanical stimulation. The BMSCs and C3H10T1/2 cells were subjected to 10% elongation for 1-7 days using a Flexcell Strain Unit, and then the mRNA levels of osteoblastic genes and the expression of core-binding factor a1 (Cbfa1) were examined. Furthermore, we focused specifically on the role of the extracellular signal-regulated kinases 1/2 (ERK1/2) and Cbfa1 in the osteogenesis of BMSCs stimulated by the stretch. The results of these experiments showed that the stretch induces a time-dependent increase in the expression of osteoblastic genes. The synthesis of osteoblastic genes was downregulated after the knockdown of Cbfa1 expression by short-interfering RNA. Furthermore, the stress-induced increase in the expression of Cbfa1 mRNA and osteoblastic genes was inhibited by U0126, an ERK1/2 inhibitor. These results indicate that long periods of intermittent traction stretch promote osteoblastic differentiation of BMSCs through the ERK1/2-activated Cbfa1 signaling pathway.”<-couldn’t get full study.

Hydrostatic pressure has been shown to induce more chondrogenic expression in MSCs when co-cultured with chondrocytes.

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture.

“Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.”<-couldn’t get the full study but this one seems to suggest that adult MSCs can upregulated chondrogenic genes by mechanical stimulation.  The details of the mechanical stimulation are left absent in the abstract unfortunately.

A combination of dynamic and shear stress has been used to induce chondrogenic differentiation in adult MSCs.

One tensile strain study at 3000 microstrain found that it upregulated both chondrogenic and osteogenic genes.

Forces induced by LSJL such as tensile strain, dynamic and shear stress, and hydrostatic pressure can induce chondroinduction of MSCs.  Whether these stimuli induce osteo- or chondro-(the ideal) induction may depend on various concentrations of growth factors in the serum(altered by supplements) and properties of the bone itself.

3 thoughts on “(Breakthrough)Direct chondroinduction by ESC implantation

  1. Lucky7

    would like to see a post of all the possible theories why it is harder to grow length vs width. Even if it is just your opinion that it will be of use. And do you think diminishing results after long periods could be lack of sensitively to chondrogenic genes or less sensitivity to hydrostatic pressure itself?

    Reply
  2. Lucky7

    you said about gene expression in the last bit, Lsjl gene changes after 32 days i think? is this a good or bad thing

    Reply

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