T cell expansion from umbilical cord blood without thymic stroma cells after stimulation with SCF, IL-7, AND IL-2

We analyzed in vitro expansion and differentiation of T progenitor cells from umbilical cord blood in the absence of thymic epithelium. The expansion setup is performed in the presence of SCF, IL-7 , and IL -2 with autologous serum .Using CBMCs as initial source , we compared the growth kinetics of several cell populations in either whole CBMC or  CD34+ -enriched-, as well as in CD3CD4CD8-depleted expansion assays by FACS analysis. After 11 days of culture, cell increase values were about 7 fold for CD3+, 6 fold for CD3+CD4+, 7 fold for CD3+CD8+, 4fold for CD3+CD56, 6fold for CD56+, and 0.2 fold for CD34+. We characterized the developmental state of these cell populations by RT –PCR analysis of the lymphoid differentiation markers RAG-1 and pre T-Alpha. In all samples , transcripts of both markers could be detected from day 0 though day 11, however , in case of pre – T-Alpha,  nested PCR  was always required , indicating lower expression . These findings; therefore, demonstrate that T-cell differentiation events (as opposed to mere expansion) do occur in stroma cell free expansion assays

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood

Background aims. Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. Methods. CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. Results. We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. Conclusions. Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.Peer reviewe

Mesenchymal Stromal Cells: Heterogeneity and Therapeutical Applications

Mesenchymal stromal cells nowadays emerge as a major player in the field of regenerative medicine and translational research. They constitute, with their derived products, the most frequently used cell type in different therapies. However, their heterogeneity, including different subpopulations, the anatomic source of isolation, and high donor-to-donor variability, constitutes a major controversial issue that affects their use in clinical applications. Furthermore, the intrinsic and extrinsic molecular mechanisms underlying their self-renewal and fate specification are still not completely elucidated. This review dissects the different heterogeneity aspects of the tissue source associated with a distinct developmental origin that need to be considered when generating homogenous products before their usage for clinical applications

Potential Application of Cord Blood-Derived Stromal Cells in Cellular Therapy and Regenerative Medicine

Neonatal stromal cells from umbilical cord blood (CB) are promising alternatives to bone marrow- (BM-) derived multipotent stromal cells (MSCs). In comparison to BM-MSC, the less mature CB-derived stromal cells have been described as a cell population with higher differentiation and proliferation potential that might be of potential interest for clinical application in regenerative medicine. Recently, it has become clear that cord blood contains different stromal cell populations, and as of today, a clear distinction between unrestricted somatic stromal cells (USSCs) and CB-MSC has been established. This classification is based on the expression of DLK-1, HOX, and CD146, as well as functional examination of the adipogenic differentiation potential and the capacity to support haematopoiesis in vitro and in vivo. However, a marker enabling a prospective isolation of the rare cell populations directly out of cord blood is yet to be found. Further analysis may help to reveal even more subpopulations with different properties, which could be useful for the directed application of these cells in preclinical models

RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells

<div><p>The amplification of RNA is becoming increasingly important, as often only limited amounts of cells are available for gene expression analysis. In this study, the gene expression profile of the 39 human homeobox (<i>HOX</i>) genes was analyzed in bone marrow-derived multipotent stromal cells (BM-MSCs) by reverse transcription (RT-) and quantitative polymerase chain reaction (qPCR). For further unlimited gene expression analysis, Whole Transcriptome Amplification (WTA) was used to amplify RNA from human BM-MSCs. However, WTA led to biased RT- and qPCR results, and even non-detectability of <i>HOX</i> transcripts compared to non-amplified BM-MSC samples which instead revealed transcription. It is important to note that the same RNA of the respective human BM-MSC line was used for normal cDNA synthesis by standard reverse transcription (non-amplified RT samples) and for cDNA synthesis by WTA (amplified WTA samples). On this account, the different RT- and qPCR results were unexpected applying WTA. The significantly reduced detection of <i>HOX</i> transcripts after WTA has been demonstrated for numerous BM-MSC lines (n = 26) by RT-PCR analysis. Furthermore, undetectable <i>HOX</i> transcripts meaning <i>HOX</i> transcripts of human BM-MSCs that were detected after normal cDNA synthesis, but were no longer detectable after WTA, were consistently observed by qPCR analysis. Finally, qPCR experiments revealed a possible explanation for the differences between amplified and non-amplified BM-MSC samples: an inverse correlation between the biased qPCR results and the low expression level of the respective <i>HOX</i> gene. The PCR analysis of high-copy transcripts like <i>GAPDH</i> or <i>RPL13A</i> revealed unchanged qPCR results after WTA compared to corresponding non-amplified BM-MSC samples. In contrast, WTA led to biased qPCR results for medium-copy <i>HOX</i> transcripts, and even non-detectability of low-copy <i>HOX</i> transcripts of human BM-MSCs resulting in false negative RT- and qPCR data applying WTA.</p></div

Expression level of <i>HOX</i> genes in human bone marrow-derived multipotent stromal cells (BM-MSCs).

<p>The expression level of seven medium-, and low copy <i>HOX</i> transcripts is documented for biological and technical replicates of BM-MSC lines (n = 3) by 2^-ΔCT value related to <i>GAPDH</i>. (A) <i>HOX</i> genes are weakly expressed in human BM-MSCs compared to high-copy transcripts like <i>RPL13A</i> showing an expression level of about 10⁻¹ related to <i>GAPDH</i>. (B) The medium-copy <i>HOX</i> transcripts present an expression level of about 10⁻³ related to <i>GAPDH</i>. (C) The expression level of low-copy <i>HOX</i> transcripts decreases to 10⁻⁴ related to <i>GAPDH</i>.</p

Overview of experimental structure.

<p>Human bone marrow-derived multipotent stromal cell (BM-MSC) lines were cultivated and expanded until passage 3 or 4. Following the RNA was isolated and the cDNA preparation was performed by standard reverse transcription and by Whole Transcriptome Amplification (WTA). The same RNA of the respective human BM-MSC line was applied for standard reverse transcription and WTA to compare RT- and qPCR results of the non-amplified and amplified cDNA samples. To exclude RNA degradation, the RNA integrity number (RIN) was determined by Agilent Bioanalyzer and only RNA samples with RIN values ≥ 9.8 were applied. Additionally, various amounts of RNA and cDNA were tested as starting material for WTA or for RT-PCR analysis.</p

Biased qPCR results for medium-copy <i>HOX</i> transcripts after Whole Transcriptome Amplification (WTA).

<p>Similar to RT-PCR analysis, the same cDNA of the respective human bone marrow-derived multipotent stromal cell (BM-MSC) line (biological triplicates, n = 3) synthesized by standard reverse transcription (RT samples) and Whole Transcriptome Amplification (WTA samples) was used for qPCR analysis. (A-C) The WTA samples revealed a significantly decreased expression level of the medium-copy <i>HOX</i> genes like <i>HOXA9</i>, <i>HOXB7</i> and <i>HOXD8</i> in comparison to RT samples. (D) The reference gene <i>RPL13A</i> is expressed on a significantly higher level compared to the <i>HOX</i> genes resulting in no significantly different qPCR results after WTA.</p

Non-detectability of <i>HOX</i> transcripts after Whole Transcriptome Amplification (WTA).

<p>In this experiment, the same RNA of the respective human bone marrow-derived multipotent stromal cell (BM-MSC) line (biological triplicates, n = 9) was applied for preparation of cDNA by standard reverse transcription (RT samples) and by Whole Transcriptome Amplification (WTA samples). (A) The so-called <i>HOX</i>-code of BM-MSCs is characterized by predominant gene expression of <i>HOXA</i>-, <i>HOXB</i>- and <i>HOXC</i>-cluster compared to <i>HOXD</i>-cluster. (B) After WTA, a significantly reduced detection of <i>HOX</i> transcripts was observed for all tested BM-MSC lines.</p

Different dilutions of amplified cDNA as starting material for RT-PCR analysis.

<p>The same cDNA from human bone marrow-derived multipotent stromal cell (BM-MSC) line 1 and 3 was constantly diluted for RT-PCR analysis. In brackets, the dilution of amplified cDNA is outlined after high-yield Whole Transcriptome Amplification (WTA) reaction (8 h amplification). (A) Lower cDNA dilutions (1:100, 1:10) led to more undetectable <i>HOX</i> transcripts like <i>HOXA9</i> or <i>HOXA10</i> compared to the recommended 1:250 cDNA dilution. (B) Higher cDNA dilutions (1:500, 1:1000, 1:2000) also revealed a higher amount of undetectable <i>HOX</i> transcripts like <i>HOXA9</i>, <i>HOXA10</i> or <i>HOXB9</i> compared to the recommended 1:250 cDNA dilution.</p