scholarly journals Direct comparison by limiting dilution analysis of long-term culture- initiating cells in human bone marrow, umbilical cord blood, and blood stem cells

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3653-3659 ◽  
Author(s):  
R Pettengell ◽  
T Luft ◽  
R Henschler ◽  
JM Hows ◽  
TM Dexter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Long Term Culture ◽  
Limiting Dilution Analysis ◽  
Cd34 Cells ◽  
Limiting Dilution

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.

scholarly journals High Integrity and Fidelity of Long-Term Cryopreserved Umbilical Cord Blood for Transplantation

2021 ◽  
Vol 10 (2) ◽  
pp. 293
Author(s):  
Gee-Hye Kim ◽  
Jihye Kwak ◽  
Sung Hee Kim ◽  
Hee Jung Kim ◽  
Hye Kyung Hong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Clinical Applications ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Umbilical cord blood (UCB) is used as a source of donor cells for hematopoietic stem cell (HSC) transplantation. The success of transplantation is dependent on the quality of cord blood (CB) units for maximizing the chance of engraftment. Improved outcomes following transplantation are associated with certain factors of cryopreserved CB units: total volume and total nucleated cell (TNC) count, mononuclear cell (MNC) count, and CD34+ cell count. The role of the storage period of CB units in determining the viability and counts of cells is less clear and is related to the quality of cryopreserved CB units. Herein, we demonstrate the recovery of viable TNCs and CD34+ cells, as well as the MNC viability in 20-year-old cryopreserved CB units in a CB bank (MEDIPOST Co., Ltd., Seongnam-si, Gyeonggi-do, Korea). In addition, cell populations in CB units were evaluated for future clinical applications. The stable recovery rate of the viability of cryopreserved CB that had been stored for up to 20 years suggested the possibility of uses of the long-term cryopreservation of CB units. Similar relationships were observed in the recovery of TNCs and CD34+ cells in units of cryopreserved and fresh CB. The high-viability recovery of long-term cryopreserved CB suggests that successful hematopoietic stem cell (HSC) transplantation and other clinical applications, which are suitable for treating incurable diseases, may be performed regardless of long-term storage.

scholarly journals Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques

2020 ◽  
Vol 9 (6) ◽  
pp. 1670
Author(s):  
Daniela Cilloni ◽  
Jessica Petiti ◽  
Valentina Campia ◽  
Marina Podestà ◽  
Margherita Squillario ◽  
...  
Keyword(s):  
Machine Learning ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Machine Learning Techniques ◽  
Specific Gene ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.

scholarly journals Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3360-3362 ◽  
Author(s):  
Karen M. Pastos ◽  
William B. Slayton ◽  
Lisa M. Rimsza ◽  
Linda Young ◽  
Martha C. Sola-Visner
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Conditioned Media ◽  
Valuable Source ◽  
Cd34 Cells ◽  
Adult Bone

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)–derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34+ cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

Humanized Sc(Fv)2 Minibody against C-Mpl Successfully Increased Platelet Number in Monkey and Increased Engraftment of Human Umbilical Cord Blood Cells in NOD/SCID Mouse.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2322-2322
Author(s):  
Takashi Yoshikubo ◽  
Yoshihiro Matsumoto ◽  
Masahiko Nanami ◽  
Takayuki Sakurai ◽  
Hiroyuki Tsunoda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Platelet Recovery ◽  
Cynomolgus Monkeys ◽  
Cd34 Cells

Abstract Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.

High Resolution Purification and Characterization of Human Cord Blood-Derived CD34–negative SCID-Repopulating Cells with a Very Immature Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 815-815
Author(s):  
Mari Murakami ◽  
Yoshikazu Matsuoka ◽  
Ryusuke Nakatsuka ◽  
Masaya Takahashi ◽  
Tsuyoshi Nakamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cell ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Nog Mice ◽  
Cell Repopulation ◽  
Limiting Dilution

Abstract Abstract 815 We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only <10% of the total CFCs were CFU-Mix. The phenotypic and functional characterizations of these 18 Lin−CD34− cells were then further investigated by cocultures with the HESS-5 murine stromal cell line in the presence of a cocktail of cytokines, such as SCF, flt3 ligand, TPO, IL-3, IL-6, and G-CSF. After 7 days of coculture, the total number of cells was observed to expand by 20 to 30 folds, 40 to 60% of which were consisted of CD34+ cells. Next, we investigated the SRC activity of these 18 Lin−CD34− cells using NOD/Shi-scid mice. When 4×104 18 Lin−CD34− cells were transplanted using IBMI, all 4 mice were highly repopulated with human CD45+ cells, including CD19+ B-lymphoid and CD33+ myeloid cells. In addition, the level of human cell engraftment in the bone marrow (BM) ranged from 93.3 to 97.5% (median, 96.8%), at 12 weeks after the transplantation. Interestingly, the BM cells obtained from the primary engrafted NOD/ Shi-scid mice that received transplants of 1,000 to 2,000 18 Lin−CD34− cells showed a secondary repopulating capacity. Furthermore, a limiting dilution analysis demonstrated the frequency of CD34− SRCs in these 18 Lin−CD34− cells to be approximately 1/1,000. The next approach to characterize the CD34− SRCs with respect to the self-renewal potential as well as the long-term repopulating potential, was to serially analyze the kinetics of engraftment for 24 weeks in the NOD/Shi-scid/IL-2Rgcnull (NOG) mice that received transplants of 2,000 18 Lin−CD34− cells containing only 2 CD34− SRCs (estimated number). All 6 mice showed signs of human cell repopulation (0.4 to 28.9%, median 2.5%) at 5 weeks after the transplantation at the contra-lateral sites of IBMI. The repopulation rates gradually increased, and reached a high level of repopulation (47.0 to 87.9%, median 72.0%) at 18 weeks after the transplantation. These results indicated that CD34− SRCs could thus sustain a long-term human cell repopulation in NOG mice, thereby suggesting that CD34− SRCs are a distinct class of primitive HSCs in comparison to the previously reported CD34+ SRCs. Disclosures: No relevant conflicts of interest to declare.

Double-Unit Umbilical Cord Blood Transplantation Induces MHC-Matched Single-Unit Dominance with Development of Immunocompetent Lymphocytes in Recipient Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4097-4097
Author(s):  
Koichi Ito ◽  
Akira Nakano ◽  
Kyoko Ito ◽  
Ikuo Kashiwakura ◽  
Hideaki Sato
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Single Unit ◽  
T And B Cells ◽  
X Ray ◽  
Cytotoxic Cells

Abstract Abstract 4097 Background: Double-unit umbilical cord blood cell (dUCBC) transplantation has emerged as an effective strategy for improving the engraftment of umbilical cord blood stem cells in the bone marrow of recipients. Due to a lack convenient animal models, analyses of the differentiation capacity of dUCBC in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In the present study, we evaluated the characteristics of immune reconstitution induced by dUCBC transplantation in mice. Materials and Methods: Natural killer cells were depleted from female C57BL/6 (B6) [H-2b] recipient mice by intraperitoneal administration of rabbit anti-asialo GM1 polyclonal antibody 1 day before transplantation. On the following day, the lethal X-ray-irradiated B6 recipients were given transplants of three different combinations of dUCBC {group (1) GFP-Tg B6 [H-2b] and BALB/c [H-2d]; group (2) GFP-Tg B6 [H-2b] and C3H [H-2k]; group (3) BALB/c [H-2d] and C3H [H-2k]}, each combination containing an equal number of cells. At 16 weeks after transplantation, reconstitution of immune cells was evaluated by flow cytometric analysis utilizing specific antibodies against lineage markers such as CD3 (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes). The donor origin of each lineage population was determined by anti-H-2Kk (for C3H) and/or H-2Kd (for BALB/c) antibody staining. GFP+ lineage cells were identified as being of B6 donor origin. Skin grafting was then performed in all recipients to assess the functional maturity of the newly developed T and B cells induced by dUCBC transplantation. Results: The survival rate at 16 weeks after transplantation was 73% (8/11) for case (1), 92% (12/13) for case (2), and 50% (3/6) for case (3). In the great majority of cases (1) and (2), in which dUCBC were administered as a stem cell source, the MHC-matched single unit from GFP-Tg B6 acts as the sole source of long-term hematopoiesis (75% (6/8) for case (1); 100% (12/12) for case (2)). CD3+ T cell peripheral blood chimerism from BALB/c was observed in two of the eight B6 survival recipient mice in case (1) at an early stage of hematopoiesis, predicting the long-term engrafting unit. On the other hand, hematopoiesis in case (3) with fully allogenenic dUCBC transplantation was reconstituted by the B6 recipients' own X-ray-resistant hematopoietic stem cells (HSC). Our results indicate that MHC-matched UCBC-HSC predominantly engraft in the recipient's bone marrow after dUCBC transplantation. However, the nature of this selective mechanism remains largely unknown. In all cases, alloreactive cytotoxic cells in recipient may participate in such selection. In dUCBC transplantation, the included allogeneic cells probably act as stimulators for promoting the differentiation and maturation of MHC-matched HSC through activation of certain types of signal transduction (for example, through cytokine secretion). Currently, we are investigating the possible presence of alloreactive cytotoxic cells in bone marrow. Functionally, these recipients were tolerant of skin grafted from B6, whereas they rejected skin from BALB/c and C3H within 20 days, indicating that both CD4+ helper and CD8+ killer T cells were functionally mature in the recipient mice. Correspondingly, only the alloantibody to BALB/c and C3H was produced in the recipients. One of two chimeric recipient mice in case (1) reacted to only C3H skin with T and B cells. Conclusions: dUCBC transplantation clearly rescued mice that had been subjected to lethal X-ray irradiation. Furthermore, our observations indicate that T and B cells derived from dUCBCs transplants are immunologically fully competent with the ability to distinguish self from non-self MHC antigens. However, a clear understanding of the mechanisms underlying the predominant engraftment of MHC-matched HSCs in the recipient's bone marrow will be necessary. Disclosures: No relevant conflicts of interest to declare.

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