Friday, October 30, 2009

Hematopoietic stem cells into chondrocytes

Hematopoietic stem cells give rise to osteo-chondrogenic cells.

"To test the ability of hematopoietic stem cells (HSCs) to give rise to osteo-chondrogenic cells, we used a single HSC transplantation paradigm in uninjured bone and in conjunction with a tibial fracture model. Mice were lethally irradiated and transplanted with a clonal population of cells derived from a single enhanced green fluorescent protein positive (eGFP(+)) HSC. Analysis of paraffin sections from these animals showed the presence of eGFP(+) osteocytes and hypertrophic chondrocytes. To determine the contribution of HSC-derived cells to fracture repair, non-stabilized tibial fracture was created. Paraffin sections were examined at 7days, 2weeks and 2months after fracture and eGFP(+) hypertrophic chondrocytes, osteoblasts and osteocytes were identified at the callus site. These cells stained positive for Runx-2 or osteocalcin and also stained for eGFP demonstrating their origin from the HSC. Together, these findings strongly support the concept that HSCs generate bone cells and suggest therapeutic potentials of HSCs in fracture repair."

"transplanted marrow cells that had been transduced with GFP-expressing retroviral vector and observed a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of the recipient mice."

" Recent studies have also identified a population of circulating human osteoblastic cells which expresses osteocalcin or alkaline phosphatase and increases during pubertal growth and during fracture repair. Studies also showed that these osteocalcin positive cells were able to form mineralized nodules in vitro and bone in vivo. This population was subsequently shown to be CD34+, suggesting that it is derived from the HSC."

"In the early process of fracture healing, a hematoma is formed and an inflammatory response occurs at the fracture site within the first 48 h, as demonstrated by the invasion of macrophages, polymorphonuclear leukocytes, and lymphocytes. Osteo-chondrogenic progenitor cells are also recruited to the fracture site within the first week after fracture. Analysis of images taken from within the fracture callus of clonally engrafted animals 7 days after fracture shows a large infiltration of eGFP+ cells. At this stage of fracture repair, the eGFP-expressing cells within the fracture callus are an unorganized, mixed population as demonstrated by their heterogeneous morphology."

"HSCs generate cells with the morphological characteristics of osteoblasts and chondrocytes in the fracture callus 2 weeks after fracture."

"with ossification, both HSC-derived (GFP+) and resident (GFP−) osteoprogenitors occupied the spaces left by apoptosis of hypertrophic chondrocytes. The slow turnover of osteoprogenitors may be one of the mechanisms of the long-term contribution of HSCs to fracture healing."

MSCs have less cell proliferation than HSCs.

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets.

"After expansion in culture, bone marrow–derived MSCs express the surface markers CD29, CD73, CD90, CD105, CD106, CD140b, and CD166, but not CD31, CD45, CD34, CD133, or MHC class II."

"nly adipose tissue–derived MSCs express high levels of CD34, and only amnion-derived MSCs are positive for stage-specific embryonic antigen (SSEA)-4 and tumor rejection antigen (TRA)-1–81. In contrast, bone marrow–derived MSCs, but not placenta-derived MSCs, express CD271 as well as tissue-nonspecific alkaline phosphatase (TNAP)."

CD9 which is downregulated in LSJL is expressed in the synovial membrane.  NT5E(CD73) is a surface marker for bone marrow and it is upregulated in LSJL.  VCAM1(CD106) is a surface marker for bone marrow and umbilical cord and is downregulated by LSJL.

Other Surface Markers altered by LSJL:
CD8b1{down}{precursor to natural killer cell according to KEGG pathway}
CD3D{down}{precursor to immune cells}
MS4A1(CD20){down}{B cell marker}
ITGA4(CD49D){down}{platelet specific marker}
CD14{up}{was also upregulated by axial loading}{monocyte lineage specific marker}

Several more stem cell surface markers were altered by LSJL than by axial loading.  LSJL seems to be an immunosuppresant. 

Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach.

"The colony-forming unit-fibroblast frequency was obtained 54 ± 1.33 from 10(3) UC-MSCs at passage 3, and the doubling time was (24.15 ± 0.49) h. Almost 10(10) UC-MSCs were largely produced in about 30 days. By flow cytometry analysis, the adherent cells displayed an abundant presence of CD73{up}, CD90 and CD105 and absence of CD34, CD45 and HLA-DR. When cultured in differentiation media, they can be differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their multidifferentiation related genes were positive. Moreover, stem cell-related transcription factors Nanog, Oct-4 and Sox-2 were positively expressed in UC-MSCs. On the basis of these findings, the single enzyme method is a good method to obtain large-scale production of MSCs from whole human UC in a short time, and the UC can be considered as a novel and convenient source of adult MSCs displaying high expansion potential and primitive pluripotent stem cells"

"To examine chondrogenic differentiation of UC-MSCs, 4 × 105 cells were created as micromass pellets and placed in cube in chondrogenic medium composed of high-glucose DMEM supplemented with 1% ITS, 10 mM l−1 ascorbate-2-phosphate, 10−7 mol l−1 dexamethasone and 10 ng ml−1 TGF-β1. Medium changes were made two to three times per week, and on day 21 of differentiation, cell pellets were harvested, fixed with 10% formalin and then finally embedded in paraffin and stained with 3% Alcian Blue (Sigma). Chondrocyte-specific genes were analysed by RT-PCR, and ACAN and collagen II were detected only in the chondrogenesis-induced cells"

"Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed.
Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73{up}, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers).
It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro."

"we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC."

"haematopoietic stem cells may trans-differentiate into epithelial and also mesenchymal lineage cells"

"Osteoblasts, osteocytes and chondrocytes were exclusively ALPP– in bones of BMT rats until the end of study, i.e. 6 months after BMT "

"The failure to detect any ALPP+, BM-derived mesenchymal lineage cells in bone or striated and smooth muscle of BMT rats irrespective of the time after BMT prompted us to ask the question whether stromal precursor cells engraft after BMT with unfractionated BM."

"haematopoietic BMC do not contribute to physiological regeneration of muscle, bone, cartilage or epithelial tissues."

"mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs."

It seems that hematopoietic stem cells were downregulated and translation into mesenchymal stem cells via Sox9 was initiated in LSJL.

"HSCs have been found preferentially localized in the endosteal region"

"we immunostained femoral sections of Nes-Gfp transgenic mice for haematopoietic lineage markers (anti-Ter119, Gr-1, CD3e, B220 and Mac-1), CD48 and CD150. In agreement with previous studies, CD150+CD48−Lin− HSCs represented a very rare subset (~0.005%) of bone marrow nucleated cells"

Hematopoietic stem cell origin of connective tissues.

"transplantation of 3000 side population cells that are highly enriched for HSCs generated osteoblasts in vivo. Transplanted marrow cells that had been transduced with GFP-expressing retroviral vector and observed a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of the recipient mice."

"Human bone marrow mesenchymal progenitor cells that are capable of adipogenic, osteogenic, and chondrogenic differentiation in culture were shown to express CD13"

"human mesenchymal progenitor cells have been identified from both CD13-expressing bone marrow cells and CD14{up}-positive peripheral blood cells"

CD34 is a hematopoietic specific marker.  LSJL upregulates laeverin as 4833403I15Rik which is homologous to CD13.

"Chondrogenic differentiation is pivotal in the active regulation of artery calcification. We investigated the cellular origin of chondrocyte-like cells in atherosclerotic intimal calcification of C57BL/6 LDLr(-/-) mice using bone marrow transplantation to trace ROSA26-LacZ-labeled cells. Immunohistochemical costaining of collagen type II with LacZ and leukocyte defining surface antigens was performed and analyzed by high-resolution confocal microscopy. Chondrocyte-like cells were detected in medium and advanced atherosclerotic plaques accounting for 7.1 +/- 1.6% and 14.1 +/- 1.7% of the total plaque cellularity, respectively. Chimera analysis exhibited a mean of 89.8% LacZ(+) cells in peripheral blood and collagen type II costaining with LcZ revealed an average 88.8 +/- 7.6% cytoplasmatic LacZ(+) evidence within the chondrocyte-like cells. To examine whether hematopoietic stem cells contribute to the phenotype, stem cell marker CD34 and myeloid progenitor-associated antigen CD13 were analyzed. CD34(+) was detectable in 86.9 +/- 8.1% and CD13(+) evidence in 54.2 +/- 7.6% of chondrocyte-like cells, attributable most likely because of loss of surface markers during transdifferentiation. Chondrocyte differentiation factor Sox-9 was detected in association with chondrocyte-like cells, whereas Sm22alpha, a marker for smooth muscle cells, could not be demonstrated. The results show that the majority of chondrocyte-like cells were of bone marrow origin, whereas CD34(+)/CD13(+) myeloid precursors appeared to infiltrate the plaque actively and transdifferentiated into chondrocytes-like cells in the progression of atherosclerosis."

"adult pluripotent cells derived from human peripheral blood monocytes showed transdifferentiation from a monocyte-like structure to a chondrocyte-like structure, indicating that these myeloid cells have the potential to differentiate into collagen type II synthesizing chondrocytes."

"bone marrow derived myeloid CD34+/CD13+ precursors actively infiltrate the plaque where they are capable of transdifferentiating into chondrocytes-like cells in the progression of atherosclerosis."

"Deep cartilage defects are repaired by precursor or stem cells of mesenchymal origin from the subchondral bone marrow. Multipotent mesenchymal stem cells from bone marrow migrate into the defect and differentiate into chondrocyte-like cells with enormous synthesis of proteoglycans and collagen II. There is, thus far, no uniformly accepted clear and specific definitive phenotype or surface marker for the identification of mesenchymal stem cells although lack of the expression of markers including CD34, CD45, CD14{up}, and CD11b have been postulated as criteria of determination."

"use CD34, CD133, and KDR{up} as markers for circulating EPC[endothelial progenitor cells] in human subjects."

Hematopoietic stem cell origin of mesenchymal cells: opportunity for novel therapeutic approaches.

"Human peripheral blood cells that express CD14, a surface protein preferentially expressed on monocytes and macrophages, were shown to generate multiple mesenchymal lineages including osteoblasts, adipocytes, chondrocytes and myocytes in culture "

Endochondral ossification is required for haematopoietic stem-cell niche formation.

"we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation."

"CD105-Thy- populations did not form bones or niches efficiently. Thus far we have not observed marrow formation without bone"

"CD105+Thy1+ progenitors may have lost chondrocyte potential"

Osterix[Sp7] is a transcription factor necessary for endochondral ossification.

"osterix knockdown severely inhibited osteogenesis and abolished niche formation, underscoring the dependence of niche formation on the process of endochondral ossification"

"perichondrial cells and chondrocytes in the developing limb express high levels of VEGF"

"CD105+Thy- mandibular and calvarial progenitors could only form marrowless bones even 60 days after transplantation"

"CD146+ subendothelial cells residing in adult human bone marrow stroma that can generate both bone and marrow when transplanted under the skin of immunodeficient mice"

Since the HSC niche is formed post endochondral ossification, can HSCs play a role in inhibiting the formation of new growth plates?

Bioreactor expansion of human adult bone marrow-derived mesenchymal stem cells.

"we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors, including stem cell factor and interleukin-3 and -6. After 8 days of culture, total cell numbers, Stro-1(+)CD44(+)CD34(-) MSCs, and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-, 29-, and 8-fold, respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin{up in LSJL}, whereas markers associated with lineage differentiation, including osteocalcin (osteogenesis), type II collagen (chondrogenesis), and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis), were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes. The rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs."

"MSCs have been cultured alongside HSCs in spinner flasks without losing their multiple mesenchymal-lineage differentiation potentials"

The bone marrow was from aged individuals 46+.

" MSCs and HSCs, were simultaneously expanded in bioreactor culture"

"After chondrogenic induction for 28 days, the bioreactor-expanded MSCs were positive for type II collagen immunostaining; paraffin-embedded cell pellets stained with alcian blue showed that chondrogenically differentiated cells were encapsulated in the chondrocytic lacunae; TEM examinations of the cell pellets demonstrated typical features of hyaline cartilage, with randomly oriented (collagen) fibrils and (proteoglycan) granules"

" As within the bone marrow cavities, MSCs and HSCs are always in close contact and support each other through the release of various growth factors, cytokines, and chemokines."

So MSCs can undergo chondrogenic differentiation in coculture with HSCs but a pro-chondrogenic factors may be needed to overcome possible HSC chondroinhibitory effects.

This study suggests that HSCs may have a positive effect on chondrogenesis:

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells.

" For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. The presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment."

6-week old male rats were used.

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts.

"KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte-specific cis-regulatory element of the collagen alpha2(XI) gene resulted in enhancement of their chondrogenic nature.  OP9 cells, another marrow stromal cell line, derived from macrophage colony-stimulating factor-deficient osteopetrotic mice and also known to be niche-constituting cells for hematopoietic stem cells expressed chondrocyte-specific or -associated genes such as type II collagen alpha1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF-beta3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen-positive hyaline cartilage within 2 weeks in vivo.  KUM5 and OP9 cells [are in] the same subcategory of "chondroblast," that is, a distinct cell type group. These two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo."

"type II collagen α1, and cartilage oligomeric matrix protein genes were expressed in OP9 cells at more than tenfold higher levels than in 9–15c mesenchymal stem cells, KUSA-O osteo-adipogenic progenitor cells, H-1/A preadipocytes, or even KUM5 chondroblasts."

Genes significantly expressed by OP9 also upregulated by LSJL:
Syndecan 2{down}
Syndecan 4
TGFBR1 {down}
VCAM1 {downs}

Genes Absent from OP9 that are downregulated by LSJL:
Col10a1 {up}
Aggrecan {up}

"[in OP9 CELLS] BMP4, BMP6, and BMP7 enhanced the TGF-β3-induced differentiation in a manner. Unlike its effect in KUM5 cells, PDGF did not inhibit TGF-β3- and BMP4-induced differentiation"

"KUM5 cells were positive (more than tenfold compared to the isotype control) for CD9, CD105 (endoglin), Sca-1 and Ly-6C, marginal for CD106 (VCAM-1) and CD140a (PDGFRα), and negative for c-kit (CD117), Flk-1, CD31 (PECAM-1), CD34, CD144 (VE-cadherin), CD45 (leukocyte common antigen), CD49d (integrin α4), CD90 (Thy-1), CD102, CD14, Ly-6G, and CD41. OP9 cells were strongly positive for CD140a, CD106, and CD9, weakly positive for Sca-1, and negative for CD105, c-kit, Flk-1, CD31, CD34, CD144, CD45, CD49d, CD90, CD102, CD14, Ly-6C, Ly-6G, and CD41 "

"OP9 cells are differentiated chondrocytes as a default state while KUM5 cells are oligopotent mesenchymal cells that have a tendency to differentiate into chondrocytes."

"The sequence of enchondral or perichondral ossification by KUM5 and OP9 cells was as follows: deposition of homogeneous matrix surrounding the small nests of the injected cells that subsequently became positive for type II collagen and exhibited metachromasia with toluidine blue staining, trapping them in the secreted homogeneous matrix, and the appearance of small nests of isogenous chondrocytes that probably resulted from repeated cell division. At a later stage, that is, 4–8 weeks after injection, the peripheral region of the generated cartilage became ossified. Importantly, the chondrogenesis by KUM5 and OP9 cells was irreversible and reproducible, and the implanted cells never transformed into malignant cells, formed any abnormal extracellular matrices, or induced any significant inflammatory reactions. It is again noteworthy that the osteogenesis by these two different lines of cells was mediated by chondrogenesis, and it was therefore considered to be chondral ossification."

"OP9 and KUM5 cells are mainly engaged in synthesizing extracellular matrix."<-This is consistent with LSJL with overrepresenation of ECM genes.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells.

" Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]{so both HSCs and chondrocytes can be derived from a common cell line}. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells."

"tissue progenitors in the bone and bone marrow are predetermined to develop into distinct tissue lineages such as osteoblasts and endothelial cells."

No comments:

Post a Comment