In Vitro Expansion of Hematopoietic Cells for Clinical Application

1995 ◽  
pp. 215-223 ◽  
Author(s):  
Stephen G. Emerson ◽  
Bernhard O. Palsson ◽  
Michael F. Clarke ◽  
Samuel M. Silver ◽  
Paul T. Adams ◽  
...  
Keyword(s):  
Clinical Application ◽  
Hematopoietic Cells ◽  
In Vitro Expansion

Clinical Scale Enrichment and Expansion of Highly Pure CD25+Foxp3+ Regulatory T Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3718-3718
Author(s):  
Carina Conrads ◽  
Jürgen Schmitz ◽  
Mario Assenmacher ◽  
Claudia Niemand ◽  
Alexander Scheffold
Keyword(s):  
Clinical Application ◽  
Cell Separation ◽  
Foxp3 Expression ◽  
Clinical Scale ◽  
Organ Rejection ◽  
Immune Mediated ◽  
In Vitro Expansion ◽  
Magnetic Cell Separation ◽  
Effector Cytokines

Abstract Abstract 3718 CD25+Foxp3+ regulatory T cell (Treg) bear great potential to prevent or treat a variety of immune mediated diseases, including autoimmunity, organ rejection or GvHD. Currently Treg for clinical application can be separated by magnetic cell separation via the CliniMACS® Plus Instrument using CD25 enrichment plus/minus prior depletion of CD8 or CD19 positive cells. With this technology Treg can be enriched to a mean purity of about 50% and first clinical trials for prevention of GvHD show no adverse effects at all. Despite these promising results, concerns have been raised whether in the setting of organ transplantation or autoimmunity higher Treg purities and/or the in vitro expansion of Treg without loss of Foxp3+ expression are required. Therefore, we have optimized the parameters for CD25 enrichment via CliniMACS to achieve higher purity of the isolated Treg. The purity of Treg could be increased by about 20–30% resulting in an average purity of 70–80% of Foxp3+ Treg. We have also developed a protocol for the in vitro expansion of CliniMACS isolated Treg using CD3/CD28 coated MACSiBead™Particles. In the presence of Rapamycin CliniMACS isolated Treg could be expanded about 20 fold with a single round of stimulation. Importantly Foxp3+ expression was not affected by the expansion but remained constant at about 70–80%. Similarly the expression of effector cytokines by expanded Treg was greatly suppressed by Rapamycin. These data show that Treg for clinical application can efficiently be isolated with high purity via CliniMACS and subsequently be expanded in vitro without loss of Foxp3 expression. Disclosures: Conrads: Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:m: Employment. Niemand:Miltenyi Biotec: Employment. Scheffold:Miltenyi Biotec: Employment.

scholarly journals Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 64-69 ◽  
Author(s):  
A.L. Petzer ◽  
C.J. Eaves ◽  
M.J. Barnett ◽  
A.C. Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Flt3 Ligand ◽  
Stem Cell Rescue ◽  
In Vitro Expansion ◽  
Stimulating Factor

We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.

scholarly journals Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2545-2553 ◽  
Author(s):  
Ute Modlich ◽  
Jens Bohne ◽  
Manfred Schmidt ◽  
Christof von Kalle ◽  
Sabine Knöss ◽  
...  
Keyword(s):  
Cell Culture ◽  
Insertional Mutagenesis ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Stable Gene ◽  
Long Terminal Repeats ◽  
Cellular Genes ◽  
In Vitro Expansion ◽  
Vector Copy Number

AbstractRetroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.

scholarly journals Short-Term In-Vitro Expansion Improves Monitoring and Allows Affordable Generation of Virus-Specific T-Cells against Several Viruses for a Broad Clinical Application

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e59592 ◽  
Author(s):  
René Geyeregger ◽  
Christine Freimüller ◽  
Stefan Stevanovic ◽  
Julia Stemberger ◽  
Gabor Mester ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Application ◽  
Short Term ◽  
In Vitro Expansion ◽  
Broad Clinical Application

scholarly journals Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 64-69 ◽  
Author(s):  
A.L. Petzer ◽  
C.J. Eaves ◽  
M.J. Barnett ◽  
A.C. Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Flt3 Ligand ◽  
Stem Cell Rescue ◽  
In Vitro Expansion ◽  
Stimulating Factor

Abstract We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.

scholarly journals Long‐term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges

2021 ◽  
Author(s):  
Stefanie Herda ◽  
Andreas Heimann ◽  
Benedikt Obermayer ◽  
Elisa Ciraolo ◽  
Stefanie Althoff ◽  
...  
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
In Vitro Expansion

scholarly journals Anti-Müllerian Hormone and OPU-ICSI Outcome in the Mare

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2004
Author(s):  
Marion Papas ◽  
Jan Govaere ◽  
Sofie Peere ◽  
Ilse Gerits ◽  
Margot Van de Velde ◽  
...  
Keyword(s):  
Clinical Application ◽  
Intracytoplasmic Sperm Injection ◽  
Independent Predictor

Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p < 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5–2.5 µg/L) or low AMH levels (<1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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