Patent Publication Number: US-6908764-B2

Title: Recombinant human cell for bone and cartilage cell formation

Description:
This is a continuation of U.S. application Ser. No. 09/376,276, filed Aug. 18, 1999 now abandoned, which in turn claims priority on German Patent No. 198 37 438.0 filed Aug. 18, 1998. 
    
    
     Despite intensive research internationally there is only one transcription factor that is able to trigger the formation of bone-forming osteoblasts at the genetic level. The properties of that factor have been described recently under a number of names: Osf2, Aml3 or Cbfa1, it being the same factor in all cases. A transcription factor for cartilage-forming chondrocytes does not currently exist. 
     Brachyury (T) is a transcription factor belonging to the so-called T-box family. Its properties and those of the T-box family have recently been described in a number of review articles (Papaioannou &amp; Silver, 1998; Smith, 1997). Brachyury and other members of that family (Tbx1 to Tbx6) are active in early embryonic development (Herrmann et al., 1990; Kispert et al., 1994; De Angelis et al., 1995; Chapman et al., 1996). 
     Brachyury has been characterised further as a transcription factor and recently the 3D-protein structure of its DNA-binding domain, the T-box, has also been clarified (Kispert et al., 1995; Müller &amp; Herrmann, 1997). Brachyury itself is responsible for the formation and proliferation of undifferentiated precursor cells, of the mesoderm. Other members of the T-box family influence the embryonic development of the neural tube (Tbx6) (Chapman &amp; Papaioannou, 1998) and of the heart (Tbx5) (Li et al., 1997; Spranger et al., 1997). It is probable that still further members of that family are involved in the embryonic patterning of limb development (Simon et al., 1997) and of the brain (T-br1) (Bulfone et al., 1995). 
     It has been known for some years that that factor (Brachyury) plays a role in early embryonic development and that it is involved in the formation of the so-called “3rd germ layer”, the mesoderm. Complete inactivation of the Brachyury factor in mice (Greek: short tail; heterozygous mice have a short tail; another name for the factor is “T”, since the gene locus is also called “T-locus”) is lethal in early embryonic development and therefore cannot easily be analysed for further influences, for example in the formation of bone and cartilage. 
     The problem of the invention is now to provide a transcription factor having bone and cartilage cell-forming properties and a human cell for the expression of the factor. 
     The problem is now solved by a recombinant human cell of undifferentiated mesenchyma for bone and cartilage cell formation, the human cell having the ability to express Tbr-1, Brachyury or another member of the T-box family. 
     The recombinant human cell can be characterised by Tbx1, Tbx2, Tbx3, Tbx4, Tbx5 or Tbx6 as a member of the T-box family. 
     Moreover, the recombinant human cell can be characterised in that it was obtained recombinantly from a cell from the following group:
         human primary stroma cell of bone marrow,   human primary mesenchymal stem cell,   human primary articular chondrocyte,   human primary chondrocyte from the epiphyses of bones, and   primary human osteoblast.       

     The recombinant human cell can also be characterised in that the human cell was obtained by biopsy, expanded and altered recombinantly in a manner known per se for the expression of Tbr-1, Brachyury or another member of the T-box family. 
     In critical tests we have been able to show that in a mesenchymal precursor cell (C3H10T½) recombinantly-expressed Brachyury is able to trigger the formation of bone- and cartilage-forming cells, the osteoblasts and chondrocytes. That test is critical since normal parental C3H10T½ cells are stable tissue cells which, only in response to very different exogenous signals, are capable of forming in four different forms of connective tissue: muscle-forming myoblasts, bone-forming osteoblasts, cartilage-forming chondrocytes and fat-forming adipocytes. 
    
    
     
         FIGS. 1 and 2  show that Brachyury is now able to trigger the formation of osteoblasts and chondrocytes in those C3H10T½ cells: 
     
    
    
     
       FIG. 1 
     
     Histologically detectable osteoblasts were detected by means of the enzymatic activity of alkaline phosphatase, a marker gene of those cells. In in vitro culture, the osteoblasts start to mature from day 4 shortly after confluence has been reached (reaching of confluence corresponds to day 0). One week later, distinct chondrocytes stained by Alcian Blue can also be detected. 
     For the formation of chondrocytes and osteoblasts it is also essential to detect the expression of specific marker genes of both cell forms. In this case the detection was carried out by a procedure called RT-PCR (reverse transcription polymerase chain reaction (PCR)). In that process firstly cDNA is prepared from the total mRNA by reverse transcription, then by means of PCR and specific primers the cDNA for specific marker genes of the osteoblast or chondrocyte formation is amplified and then detected by gel electrophoresis. 
     
       FIG. 2 
     
     According to the invention it has been shown that highly specific marker genes for osteoblast formation, such as, for example, the osteocalcin gene and the PTH/PTHrP receptor gene, are highly regulated in Brachyury-overexpressing cells. Collagen (I) also exhibits the typically biphase course of osteoblast differentiation. Chondrocyte formation is confirmed by the early high rates of expression of collagen II; moreover it is also probable that the chondrocytes become mature since the formation of collagen (II)-mRNA from day 7 of the culture is down-regulated and other collagens are probably then expressed. 
     REFERENCES 
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     Chapman, D. L., Garvey N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M., and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206:379-390. 
     Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391:695-697. 
     De Angelis, M. H., Gründker, C. Herrmann, B. G., Kispert, A., and Kirchner, C. (1995). Promotion of gastrulation by maternal growth factor in cultured rabbit blastocysts. Cell Tissue Res. 282:147-154. 
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