scholarly journals Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3306-3313 ◽  
Author(s):  
QL Hao ◽  
FT Thiemann ◽  
D Petersen ◽  
EM Smogorzewska ◽  
GM Crooks
Keyword(s):  
Cord Blood ◽  
Hematopoietic Progenitors ◽  
Generative Capacity ◽  
Long Term Culture ◽  
Clonal Proliferation ◽  
Unit Cells ◽  
Functional Hierarchy

Long-term culture-initiating cells (LTC-IC) are hematopoietic progenitors able to generate colony-forming unit-cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on bone marrow (BM) stroma and represent the most primitive progenitors currently detectable in vitro. We have recently reported that long-term cultures initiated with CD34+CD38- cells from BM or cord blood are able to continue generating CFU for at least 100 days, ie, beyond the standard LTC-IC period. In this report, single-cell cultures from cord blood and retroviral marking of cord blood and BM were used to study whether the subpopulation of CD34+CD38- cells able to generate CFU beyond 60 days (“extended long-term culture-initiating cells” or ELTC-IC) are functionally distinct from LTC-IC in terms of timing of initial clonal proliferation and generative capacity. All cord blood LTC-IC formed clones of greater than 50 cells by day 30. In contrast, cord blood ELTC-IC proliferated later in culture, 50% forming clones after day 30. Although efficient retroviral marking of LTC-IC was seen (25% to 45%), marking of ELTC-IC was inefficient (< 1%), consistent with a more quiescent progenitor population. There was a positive correlation between time of clonal proliferation and generative capacity. ELTC-IC generated threefold to fourfold more progeny than did LTC-IC (P < .002). These studies show that there is a functional hierarchy of progenitors in long-term culture which correlates with their level of quiescence. By extending the LTC-IC assay, a more primitive progenitor may be studied that may be functionally closer to the human long-term repopulation stem cell in vivo.

Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1261-1271 ◽  
Author(s):  
DL DiGiusto ◽  
R Lee ◽  
J Moon ◽  
K Moss ◽  
T O'Toole ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Cell Cultures ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Hematopoietic Progenitors ◽  
Phenotypic Analysis

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)- 3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos

1999 ◽  
Vol 55 (3-4) ◽  
pp. 151-162 ◽  
Author(s):  
M Stojkovic ◽  
M Büttner ◽  
V Zakhartchenko ◽  
J Riedl ◽  
H.-D Reichenbach ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Long Term Culture

Distinct Requirements for Optimal Growth and In Vitro Expansion of Human CD34+CD38− Bone Marrow Long-Term Culture-Initiating Cells (LTC-IC), Extended LTC-IC, and Murine In Vivo Long-Term Reconstituting Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4093-4102 ◽  
Author(s):  
Veslemøy Ramsfjell ◽  
David Bryder ◽  
Helga Björgvinsdóttir ◽  
Sten Kornfält ◽  
Lars Nilsson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Activity ◽  
Optimal Growth ◽  
Cell Manipulation ◽  
Long Term Culture ◽  
Human Cd34

Abstract Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (&gt;60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34+CD38− phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34+CD38− BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34+CD38− progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

Retroviral Mediated Transfer of MLL Fusion Genes into Human CD34+ Cord Blood Cells Supports the Establishment of Long-Term Myeloid Cultures with Growth Rates, Immunophenotypic and Transcriptional Features Distinct from Those of AML1-ETO Transduced Cultures.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1610-1610
Author(s):  
Jorge F. DiMartino ◽  
Catherine Rettig ◽  
Mark Wunderlich ◽  
James C. Mulloy
Keyword(s):  
Transcription Factors ◽  
Cord Blood ◽  
Leucine Zipper ◽  
Antigen Expression ◽  
Fusion Genes ◽  
Human Cord Blood ◽  
Myeloid Progenitors

Abstract Translocations involving the MLL gene, primarily t(9;11) and t(10;11) together with rearrangements affecting the core binding factor (CBF) genes, t(8;21) and inv(16) comprise the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). Although all of these rearrangements generate chimeric transcription factors (MLL-AF9, MLL-AF10, AML1-ETO and CBFβ-MYH11) clinicopathologic features and transcriptional profiles clearly distinguish MLL-rearranged from CBF-rearranged AML. To understand how these distinct subgroups of AML arise, we have developed a model for studying the effects of MLL and CBF fusion proteins on the growth, survival and differentiation human myeloid progenitors in vitro. Using retroviral mediated gene transfer, we transduced CD34 selected normal human cord blood (CB) cells with vector (MIEG3) alone or with vectors expressing MLL-AF9, MLL-AF10 or AML1-ETO fusion genes. Whereas CB transduced with MIEG3 proliferated in liquid culture for 6 to 8 weeks, MLL-AF9, MLL-AF10 and AML1-ETO transduced cells have continued to proliferate continuously in culture for more than 16 weeks without any sign of crisis. At any point after transduction, CB expressing MLL-AF9 or MLL-AF10 exhibited a faster rate of growth as compared with AML1-ETO or MIEG3 transduced CB. This difference in growth rate was associated with a reduced frequency of spontaneous apoptosis by annexin staining in the MLL cultures, as compared with the AML1-ETO cultures, but no difference in the fraction of cells in S-phase. MLL-AF9 and MLL-AF10 transduced CB also exhibited evidence of early myeloid maturation arrest based on morphology and surface antigen expression. However, while AML1-ETO transduced cells continue to express CD34 throughout their time in culture, MLL cultures lose expression of this stem cell-associated antigen and acquire expression of c-Kit and CD33, neither of which is expressed in AML1-ETO cultures. Also, unlike AML1-ETO transduced cells, CB transduced with with MLL fusions retain serial clonogenicity for 3 or more rounds of plating in methylcellulose assays. We used quantitative realtime RT-PCR to measure expression of 3 genes that are differentially expressed in patients with MLL or CBF gene fusions based on published microarray data. While expression of SPARC increased over time in MIEG3 cultures or remained stable in CB transduced with AML1-ETO, it decreased to nearly undetectable levels in MLL-AF9 transduced cultures. In contrast, expression of both BMI-1 and HOXA9 increased in the MLL-AF9 cultures and decreased in the MIEG3 and AML1-ETO cultures. The transcriptional changes in our long-term cultures mirror the gene expression differences that have been observed in AML associated with MLL or CBF fusions and suggest that this will be a useful model to study how chimeric transcription factors contribute to myeloid leukemogenesis. Interestingly, CB transduced with a mutated MLL-AF10 (MA10ΔLZ) lacking the leucine zipper domain required for transformation of primary murine myeloid progenitors did not differ, in terms of growth or differentation, from MIEG3 transduced cells. This suggests that the effects of MLL-AF9 and MLL-AF10 on normal CB may reflect early events in myeloid leukemogenesis. The in vivo leukemogenic potential of MLL fusion transduced CB is currently being evaluated in NOD/SCID mice.

Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2237-2237
Author(s):  
Ravindra Majeti ◽  
Christopher Y. Park ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2444-2444
Author(s):  
Ewa K Zuba-Surma ◽  
Magdalena Kucia ◽  
Rui Liu ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia2007;21:297–303) These VSELs are: smaller than erythrocytes; SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−; responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin). Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. Figure Figure

Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1097-1105 ◽  
Author(s):  
G. Güenechea ◽  
J.C. Segovia ◽  
B. Albella ◽  
M. Lamana ◽  
M. Ramı́rez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P &lt; .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

Distinct Requirements for Optimal Growth and In Vitro Expansion of Human CD34+CD38− Bone Marrow Long-Term Culture-Initiating Cells (LTC-IC), Extended LTC-IC, and Murine In Vivo Long-Term Reconstituting Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4093-4102 ◽  
Author(s):  
Veslemøy Ramsfjell ◽  
David Bryder ◽  
Helga Björgvinsdóttir ◽  
Sten Kornfält ◽  
Lars Nilsson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Activity ◽  
Optimal Growth ◽  
Cell Manipulation ◽  
Long Term Culture ◽  
Human Cd34

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34+CD38− phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34+CD38− BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34+CD38− progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

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