Neocartilage formation from predifferentiated human adipose derived stem cells in vivo

Abstract

Aim: To examine the chondrogenic potential of human adipose derived stem cells (hASC) induced by human transforming growth factor beta2 (hTGF beta2) in vitro, and to investigate if predifferentiated hASC can produce neocartilage in vivo. Methods: hASC were isolated from subcutaneous adipose tissue and cultured in pellets with the addition of hTGF beta2. Chondrogenic differentiation was assayed by RT-PCR, Western blotting, toluidine blue staining, and immunohistochemistry staining for collagen type II. For the in vivo study, intact induced cell pellets or the released cells embedded in alginate gel with different concentrations were implanted subcutaneously in nude mice. Specimens were harvested at different time points and carried with histological and immunohistochemistry examination to evaluate the cartilage formation. Results: RT-PCR analysis revealed that hASC produced aggrecan and collagen type II after 7 d of induction and continued throughout the culture period. This was also demonstrated by the Western blot analysis, positive staining of toluidine blue, and immunohistochemistry for collagen type II. After reseeding in the monolayer, the cells isolated from the pellets displayed a polygonal morphology compared with the primary spindle shape. hASC were released from the induced cell pellets when embedded in alginate gel (implanted cell concentration=5×10 6

Chondrogenic differentiation of adipose tissue‐derived mesenchymal stem cells: Greater doses of growth factor are necessary

“…24 As demonstrated by the work of Barry et al, 1 TGF-b 2 and b 3 isoforms have stronger chondrogenic potential than TGF-b 1 . This point was also confirmed in our unpublished data.…”

Isolation, Culturing, Characterization and Aging of Adipose Tissue-derived Mesenchymal Stem Cells: A Brief Overview

“…Variable number of stem cells is present in different tissues such as liver, heart, kidney, skin, bone marrow, adipose tissue, placenta, dental, amniotic fluid, amnion and umbilical cord blood as well as in various fetal tissues (Wei et al 2000;Banas et al 2007). Earlier it was considered that stem cells can only differentiate into mature cells of the same organ but recent evidences have shown that they can also differentiate into other cell types and even into the cells of ectoderm, mesoderm and endoderm (Beltrami et al 2001;De Ugarte et al 2003;Jin et al 2007;Vieira et al 2010;Ren et al 2012). It has been demonstrated that MSCs include a heterogeneous population of cells with multilineage differentiation potential, ability to modulate oxidative stress, and produce a variety of factors that are capable of manipulating a broad range of biological functions (Caplan 2007).…”

“…The isolation method and the cytological characterization of stromal precursor cells from adipose tissue were shown at the beginning of the 1970s (Poznanski et al 1973). ADSCs have been obtained from bovine, dog, goat, horse, rabbit, rat, mouse, pig and human (Noer et al 2006;Vidal et al 2007;Jin et al 2007;Torres et al 2007;Yamamoto et al 2007;Williams et al 2008;Vieira et al 2010;Hall et al 2010;Nakanishi et al 2011;Abd El Samad 2011;Ren et al 2012;Strioga et al 2012;Ahmad and Shakori 2013). It has been published for ADSCs that liposuction side, liposuction procedure, variations in the isolation techniques, growth media, culture conditions, age or body mass index play an important role in the cell yield, growth, and frequency of stem cells causing a remarkable difference in their proliferation and differentiation capacity (Tsai et al 2006;Jurgens et al 2008;Baer et al 2012).…”

“…Adipose tissue is minced with fine scissors and surgical scalpels into less than 3 mm 3 pieces. The adipose tissue is washed extensively with sterile equal volumes of PBS or Hank's balanced salt solution (HBSS) containing antibiotics to remove the contaminating debris and red blood cells (Noer et al 2006;Jin et al 2007;Vieira et al 2010). To isolate ADSCs, the sample is spun at 2000 rpm for 5 min to collect the fat tissue pieces.…”

“…Enzyme activity is neutralized with DMEM high or low glucose containing 10% fetal bovine serum (FBS) and centrifuged at 1200 rpm for 10 min to obtain a pellet. The pellet is re-suspended in complete medium (DMEM containing 10-15% FBS, 100 U/mL penicillin, and 100 µg/mLstreptomycin) and filtered through a 100 µm nylon mesh to remove the cellular debris (Ko et al 2007;Jin et al 2007; Ahmad and Shakori 2013). The number of cells is counted by hemocytometer and the cells are allowed to adhere to a plastic culture plate at 10 4 cells/cm 2 and incubated overnight at 37ºC under 5% CO 2 .…”