Committed Stem Cells Research Articles

Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells

Evidence is presented that bone marrow (BM) in addition to CD45(positive) hematopoietic stem cells contains a rare population of heterogenous CD45(negative) nonhematopoietic tissue committed stem cells (TCSC). These nonhematopoietic TCSC (i) are enriched in population of CXCR4(+) CD34(+) AC133(+) lin(-) CD45(-) and CXCR4(+) Sca-1(+) lin(-) CD45(-) in humans and mice, respectively, (ii) display several markers of pluripotent stem cells (PSC) and (iii) as we envision are deposited in BM early in development. Thus, since BM contains versatile nonhematopoietic stem cells, previous studies on plasticity trans-dedifferentiation of BM-derived hematopoietic stem cells (HSC) that did not include proper controls to exclude this possibility could lead to wrong interpretations. Therefore, in this spotlight review we present this alternative explanation of 'plasticity' of BM-derived stem cells based on the assumption that BM stem cells are heterogenous. We also discuss a potential relationship of TCSC/PSC identified by us with other BM-derived CD45(negative) nonhematopoietic stem cells that were recently identified by other investigators (eg MSC, MAPC, USSC and MIAMI cells). Finally, we discuss perspectives and pitfalls in potential application of these cells in regenerative medicine.

PTPN11 and RAS Gene Mutation Pattern Identifies an Unique Feature of Upregulated RAS Function in Infant ALL.

Point mutations affecting RAS GTPase activity promote upregulation of RAS signaling, and are among the most common genetic alterations in human cancers. Oncogenic mutations in NRAS and KRAS2 genes are found with variable prevalence in hematological malignancies, including ALL. Upregulation of RAS signaling can also results from molecular lesions in genes coding transducers controlling RAS function. We recently provided evidence that PTPN11 is mutated in approximately one-third of pediatric ALL cases. Significantly, while PTPN11 mutations appeared to be preferentially associated with the common immunophenotype, mutations in NRAS or KRAS2 were uniformly distributed among the B-cell precursor ALL subtypes. Very few are the data on RAS pathway status in pediatric ALL patients aged <1 year (Infant). Infant ALL is a rare subgroup of leukemia peculiar for several characteristics, mainly related to a possible origin from a not-fully committed stem cell. In order to explore whether alterations of RAS signaling may identify specific subgroups within this heterogeneous patient population, we analyzed 87 Infant cases prospectively enrolled in the Interfant-99 International ALL protocol. This is the largest series of Infant ALL analyzed for alteration of the RAS pathway. PTPN11 (exon 3 and 13), NRAS (exon 1 and 2), and KRAS2 (exon 1 and 2) mutation analysis was performed by DHPLC analysis and direct sequencing. None of the 87 Infant cases carried a PTPN11 somatic mutation. By contrast, a high prevalence of mutations affecting NRAS and KRAS2 was observed (approximately 30%). Thirteen cases (14.9%) were positive for a mutation in exon 1 (codons 12 and 13, 10 cases, 11.5%) or exon 2 (codon 61, 3 cases, 3.4%) of the NRAS gene. Seventeen cases (19.5%) carried a variety of exon 1 KRAS2 mutations: codons 12 and 13, n=13 (14.9% of total); insAGC(30–32), n=1; G(40)C, n=1; C(53)A, n=1; G(57)C, n=1. Interestingly, in 9/27 cases (33.3% of RAS mutated cases, 10.3% of total) DHPLC and sequencing consistently showed the presence of the RAS mutation only in a fraction (10 to 40%) of the blast cells population. In one additional case a KRAS2 mutation was observed in a subset of cells along with a full NRAS mutation. In two more cases mutations affecting NRAS and KRAS2 coexisted on the same patient; in both cases the mutations were representative of a subgroup of the total cell population. In conclusion: differently from older children, PTPN11 gene mutations are a rare event in infant ALL. This could be related to the low incidence of the “common” immunophenotype in Infants. By contrast, the overall incidence of RAS gene mutations is high, accounting for about 30% of cases. Interestingly, in 10% of cases a mutation (or even more than one) was found in a fraction of the blast cell population. This could reflect the fact that RAS mutations were secondary to other genetic events, and in any case related to the oligoclonal characteristic of this age-specific ALL subgroup. The prognostic effect of these genetic events must be evaluated in a longer follow up time of prospective series of patient, as in the Interfant Consortium.

More Evidence That the Phenomenon of “Plasticity” of Hematopoietic Stem Cells (HSC) and Potential Involvement of BM-Derived Cells in Organ Regeneration Could Be Explained by the Presence of a Heterogenous Population of Tissue Committed Stem Cells (TCSC) Residing/Hiding out in Bone Marrow.

Recently we identified a mobile, SDF-1 responsive population of CXCR4+ bone marrow mononuclear cells that express mRNA for various markers of early tissue committed stem cells (TCSC) in bone marrow (BM) by employing chemotactic isolation to an SDF-1 gradient combined with real time RT-PCR and immunohistochemical analysis (Leukemia 2004: 18;29-40). In the current work we report new data showing that murine and human TCSC (i) are highly mobile and respond robust to HGF and LIF gradients, in addition to an SDF-1 gradient, (ii) are released (mobilized) from BM into peripheral blood during organ injury (e.g., stroke, heart infarct, partial body irradiation), (iii) are highly enriched in mRNA for pluripotent stem cell transcription factors such as Oct-4, Rex-1 and Nanog, iv) reside in a population of BM-derived Sca-1+ (mice) and CD34+AC133+ (human) non-hematopoietic CD45 negative cells, v) are small (~7 mm in diameter), vi) contain supercoiled DNA, vii) rapidly adhere to/undergo emperipolesis in fibroblasts and thus are enriched in a fraction of BM-derived adherent cells, viii) their number is highest in BM from young (1–2 month-old) mice and decreases in 1-year-old animals, and similarly ix) these cells are significantly diminished in short living DBA/2J mice as compared to long living B6 mice. Thus, these findings further support our theory of the BM as a “hideout” of TCSC and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as trans-dedifferentiation/plasticity of HSC. Since we noticed that not only SDF-1 but also HGF and LIF are upregulated in damaged tissues (e.g., brain, heart or liver), we postulate that TCSC that express CXCR4, c-Met, and LIF-R after being mobilized from the BM into PB, may be subsequently chemoattracted to damaged organs where they play a role in tissue repair/regeneration. Moreover, our observation that the number of TCSC is the highest in BM of young animals and decreases with age and is significantly lower in short-living murine strains provides a novel insight into aging and may explain why the regeneration process becomes less effective in older individuals. Based on our observation, we conclude that BM-derived HSC are not plastic but BM in addition to HSC also contains heterogenous populations of TCSC.

Delayed haematological complications of mustard gas

Haematopoiesis could be affected by mustard gas. We randomly selected 318 chemical victims exposed to mustard gas and compared their cell blood counts and peripheral blood smears (PBS) with those of 377 healthy men, and also various haematological indices of 57 of these victims compared with previous data 5 years ago. The average number of red blood cells and haemoglobin of victims compared with the controls was not significantly different, but they were increased compared with data from 5 years ago. White blood cell counts, neutrophils and lymphocytes did not show any clinically meaningful difference compared with the control group but 20 cases with atypical lymphocytes in their PBS have been found. Change in lymphocyte shape may be related to committed stem cell involvement. Further studies on bone marrow cells and cell markers are needed to document this hypothesis. The mild increase in erythroid cells and haemoglobin concentration may be due to chronic obstructive pulmonary disorder and other respiratory diseases in these patients.

Neutrophil Function in Preterm and Term Infants

After completing this article, readers should be able to: 1. List the primary cell functions of circulating neutrophils in response to microbial invasion and inflammation. 2. Describe the interaction between neutrophils and endothelial cells and how these interactions differ between neonatal and adult cells. 3. Describe differences in adhesion of phagocytes between neonatal and adult neutrophils. 4. Describe the phagocytosis-associated respiratory burst and deficiencies seen in neonatal neutrophils. Polymorphonuclear neutrophils (PMN) constitute the primary line of defense in the cellular immune system. Inborn defects in the generation of neutrophils or in neutrophil function are predisposing risk factors for life-threatening infections in infants and children. In general, newborns have an increased risk for systemic infections, and very immature preterm infants are especially prone to developing nosocomial bloodstream infections that clearly have been associated with increased morbidity and mortality. In this review, we attempt to identify possible quantitative and qualitative deficiencies observed in neonatal neutrophils and try to define, whenever possible, the underlying mechanisms of neutrophil dysfunctions in term and preterm infants. (1) ### Bone Marrow Storage Pool and Circulating Pool Neutrophils and cells of the mononuclear-phagocyte system originate in the bone marrow as a common committed progenitor cell for the granulocyte and the monocyte-macrophage pathways: the colony-forming unit-granulocyte monocyte (CFU-GM). Glycoprotein hormones termed colony-stimulating factors (G-CSF/GM-CSF) induce proliferation, maturation, and differentiation into neutrophils and monocytes. (1) The sequential development of neutrophilic granulocytes proceeds from pluripotent progenitor cell to committed stem cell to myeloblast to promyelocyte to myelocyte to metamyelocyte to band to segmented neutrophil. This sequence occurs within the bone marrow and possibly is regulated by the interaction of the hematopoietic inductive environment, CSF, and specific inhibitors of granulopoiesis. (2) Myeloblasts, promyelocytes, myelocytes, and other cells of the CFU-GM possess the possibility of cell division and form, by definition, the proliferative pool. Rodent studies have shown clearly that the total …

HIV-1 protease inhibitor induces growth arrest and apoptosis of human multiple myeloma cells via inactivation of signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2

We previously showed that HIV-1 protease inhibitors slowed the proliferation of human myeloid leukemia cells and enhanced their differentiation in the presence of all-trans retinoic acid (ATRA). In this study, we found that protease inhibitors, including ritonavir, saquinavir, and nelfinavir, but not indinavir, induced growth arrest and apoptosis of U266, RPMI8226, and ARH77 human multiple myeloma (MM) cells in association with down-regulation of antiapoptotic protein Mcl-1. Also, protease inhibitors inhibited the survival of freshly isolated MM cells from patients. In contrast, these protease inhibitors did not affect survival of normal B cells and colony formation of myeloid committed stem cells (CFU-GM) from healthy volunteers. In addition, we found that all of the protease inhibitors, except for indinavir, blocked interleukin-6 (IL-6)-stimulated phosphorylation of both signal transducer and activator of transcription 3 (STAT 3) and extracellular signal-regulated kinase 1/2 in U266 and RPMI8226 MM cells. Moreover, the protease inhibitors inhibited both the basal and IL-6-stimulated STAT 3/DNA binding activity in U266 cells as measured by an ELISA-based assay. Furthermore, ritonavir inhibited production of vascular endothelial growth factor one of the targets of STAT 3, in U266 and RPMI8226 cells as measured by ELISA. Taken together, protease inhibitors might be useful for treatment of individuals with MM.

Skin: a promising reservoir for adult stem cell populations

Plasticity of adult cells has been identified in several post-natal tissues in the past few years and has attracted special attention in regenerative medicine. Skin is the biggest organ in the body. Adult skin consists of epidermis, dermis and appendages such as hairs and glands which are linked to the epidermis but project deep into the dermal layer. Skin stem cell biology has been a focus of increasing interest in current life science. Committed stem cells with limited differentiation potential for regeneration and repair of epidermis have been known for decades. Recent studies further report that adult skin tissues contain cell populations with pluripotent characteristics. Multipotent stem cells from hair follicle and non-follicular skin, both in epidermal and dermal tissues, are found to have the differentiation capacity to generate multiple cell lineages. Basing on the present data, our hypothesis is that skin may serve as a local reservoir of various adult stem cell populations, including committed stem cell populations and pluripotent stem cell populations both in epidermal and dermal tissues. Given its easy accessibility, stem cells in skin will not only provide an experimental model for skin biology, but also may provide an experimental model for studying the epithelial–mesenchymal interactions of several other organs outside of skin. The stem cell populations in skin tissues may also have extensive therapeutic implications in the replacement of skin and may serve as an alternative source of stem cells for several other organs outside of skin. The in situ activation and mobilization of stem cell populations in the skin is an ideal way to renew and repair epidermis and dermis, even appendages.

Embryonic committed stem cells as a solution to kidney donor shortage

The number of human kidney transplants has increased rapidly in recent years, but the need greatly exceeds organ availability. Induction of appropriate kidney differentiation and growth from stem or progenitor cell populations represents an attractive option to combat chronic kidney donor shortage. In an analogy to haematopoietic stem cells, which are much more efficient in giving rise to blood than to other cell types, if any at all, renal stem cells could afford an unlimited source for regenerating nephrons. While a single nephrogenic stem cell has not been characterised, indirect evidence suggests that a renal stem cell population is contained within the metanephric mesenchyme, which along with a branch of the Wolffian duct represents the direct precursor of the mature kidney. Human tissue fragments derived from these developing precursors can regenerate renal structures when grafted into mice. Moreover, recent data pinpoints a window of time in human and pig kidney development that may be optimal for transplantation into mature recipients. ‘Window’ transplants are defined by their remarkable ability to grow, differentiate and undergo vascularisation, achieving successful organogenesis of urine-producing miniature kidneys with no evidence of transdifferentiation into non-renal cell types, lack of tumourigenicity and reduced immunogenicity compared with adult counterparts. In contrast, ‘non-window’ transplants (earlier or later in gestation) can form teratomas or are more prone to immune rejection and are less suitable for organogenesis. Hopefully, the use of stage-specific early human and porcine kidney precursors to cultivate mature kidney cells in vivo, possibly in conjunction with other modalities of stem cell technology and tissue engineering, will prove valuable to sustain life in patients with failing kidneys.

Embryonic committed stem cells as a solution to kidney donor shortage

Embryonic committed stem cells as a solution to kidney donor shortage

PC-SPES decreases proliferation and induces differentiation and apoptosis of human acute myeloid leukemia cells

PC-SPES is an eight herbal mixture which has been shown to be active against prostate cancer cells in vitro as well as in patients. In this study, we discovered that it has anti-leukemia activity. HL-60, NB4, U937 and THP-1 human acute myeloid leukemia cells were cultured in the presence of various concentrations of PC-SPES (0.06-0.5 micro l/ml) for 4 days, and cell numbers were counted by Trypan blue exclusion. PC-SPES inhibited proliferation of these cells with an ED50 of 0.17, 0.09, 0.18, 0.32 micro l/ml, respectively. In clonogenic assay, PC-SPES inhibited growth of HL-60 cells (ED50, 0.043 micro l/ml). On the other hand, PC-SPES (0.1 micro l/ml) stimulated growth of normal myeloid committed stem cells (CFU-GM) by 1.4-fold of control (p=0.03). Anti-leukemia effects also occurred against freshly isolated leukemia cells from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. Interestingly, when PC-SPES was combined with ATRA, the antiproliferative effect was markedly enhanced. For example, PC-SPES (0.125 micro l/ml) or ATRA (10(-8) mol/l) inhibited growth of HL-60 cells after 4 days of culture, by approximately 40 and 30%, respectively; simultaneous treatment with both, suppressed growth by 80%. In addition, PC-SPES induced differentiation of HL-60 and NB4 cells, as measured by expression of CD11b and reduction of NBT. ATRA synergistically enhanced this activity. For example, either PC-SPES (0.5 micro l/ml) or ATRA (10(-8) mol/l) induced 23 and 18% of HL-60 cells, respectively to express CD11b on day 2 of culture; and when both were combined, 60% of HL-60 cells were stimulated to express CD11b antigen. Furthermore, PC-SPES (0.5 micro l/ml) produced apoptosis of HL-60 and NB4 cells, as measured by TUNEL assay, with 17% of HL-60 cells and 52% of NB4 cells becoming apoptotic on their third day of culture. Importantly, PC-SPES stimulated expression of the novel myeloid specific transcription factor C/EBPepsilon in HL-60 and NB4 cells. Taken together, PC-SPES inhibits growth and induces differentiation and apoptosis of myeloid leukemia cells, and enhances the antiproliferative and prodifferentiative effects of ATRA on these cells. PC-SPES might be useful with ATRA for treatment of patients with acute promyelocytic leukemia (APL), and it could have a role in other types of cancers including MDS.

Common Adult Stem Cells in the Human Breast Give Rise to Glandular and Myoepithelial Cell Lineages: A New Cell Biological Concept

Breast biology and pathology are currently shaped by the two-cell concept that recognizes only glandular and myoepithelial cells. In the present study, we have visualized a previously unidentified cell population within the epithelial compartment of the breast, which displays the phenotypic characteristics of a committed stem cell.Immunofluorescence double labeling with digital image processing and Western blotting were applied to normal breast tissue as well as to noninvasive and invasive breast cancers using antibodies to basal cytokeratin 5 (Ck5), glandular cytokeratins 8/18 (Ck8/18/19), and smooth muscle α-actin (SMA) as markers for myoepithelial cells (SMA).A distinct population of cells was identified that expressed Ck5 in the absence of Ck8/18/19 or SMA. These cells differentiate toward glandular epithelial or myoepithelial Ck5-negative end cells passing through either Ck5/Ck8/18/19 or Ck5/SMA-positive intermediates. Our experiments clearly demonstrate a precursor or committed stem cell function of the Ck5-positive cell that is responsible for regeneration of the human adult breast epithelium. However, the observation that the vast majority of breast cancers display the glandular epithelial immunophenotype strongly suggests that the neoplastic cells derive from a late stage of the glandular epithelial differentiation pathway. The significance of this new cell biological model is that it might serve as a tool to unravel the regulatory mechanisms that govern regeneration and abnormal proliferation of breast epithelium at the cellular level.

Neural induction, neural fate stabilization, and neural stem cells.

The promise of stem cell therapy is expected to greatly benefit the treatment of neurodegenerative diseases. An underlying biological reason for the progressive functional losses associated with these diseases is the extremely low natural rate of self-repair in the nervous system. Although the mature CNS harbors a limited number of self-renewing stem cells, these make a significant contribution to only a few areas of brain. Therefore, it is particularly important to understand how to manipulate embryonic stem cells and adult neural stem cells so their descendants can repopulate and functionally repair damaged brain regions. A large knowledge base has been gathered about the normal processes of neural development. The time has come for this information to be applied to the problems of obtaining sufficient, neurally committed stem cells for clinical use. In this article we review the process of neural induction, by which the embryonic ectodermal cells are directed to form the neural plate, and the process of neural�fate stabilization, by which neural plate cells expand in number and consolidate their neural fate. We will present the current knowledge of the transcription factors and signaling molecules that are known to be involved in these processes. We will discuss how these factors may be relevant to manipulating embryonic stem cells to express a neural fate and to produce large numbers of neurally committed, yet undifferentiated, stem cells for transplantation therapies.

PPARγ-dependent and PPARγ-independent effects on the development of adipose cells from embryonic stem cells

Peroxisome proliferator-activated receptor (PPAR) γ was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARγ in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARγ is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARγ in developing EBs over-expressing PPARγ did not commit stem cells towards the adipose lineage. In differentiated PPARγ −/− EBs, only markers characteristic of preadipocytes were found to be expressed. PPARδ is present in EBs but did not compensate for the lack of PPARγ in terminal differentiation. Taken together, these results favor a critical PPARγ-independent phase culminating in preadipocyte formation that precedes a PPARγ-dependent phase in the development of adipose cells from pluripotent stem cells.

Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation

Muscle satellite cells are believed to represent a committed stem cell population that is responsible for the postnatal growth and regeneration of skeletal muscle. However, the observation that cultured myoblasts differentiate into osteocytes or adipocytes following treatment with bone morphogenetic proteins (BMPs) or adipogenic inducers, respectively, suggests some degree of plasticity within the mesenchymal lineage. To further investigate this phenomenon, we explore the osteogenic and adipogenic potential of satellite cells isolated from adult mice. Our experiments clearly demonstrate that satellite cell-derived primary myoblasts, expressing myogenic markers such as MyoD, Myf5, Pax7 and desmin, differentiated only into osteocytes or adipocytes following treatment with BMPs or adipogenic inducers, respectively. However, satellite cells on isolated muscle fibers cultured in Matrigel readily differentiated into myocytes as well as osteogenic and adipogenic lineages, whereas primary myoblasts did not. Satellite cell-derived primary myoblasts isolated from mice lacking the myogenic transcription factor MyoD ( MyoD-/-) differentiate into myocytes poorly in vivo and in vitro (Megeney et al., Genes Dev. 1996; Sabourin et. al, J. Cell Biol., 1999). Therefore, we tested whether MyoD-/- primary myoblasts display increased plasticity relative to wild type cells. Unexpectedly, the osteogenic or adipogenic differentiation potential of MyoD-/- primary myoblasts did not increase compared to wild-type cells. Taken together, these results strongly suggest that muscle satellite cells possess multipotential mesenchymal stem cell activity and are capable of forming osteocytes and adipocytes as well as myocytes.

Ca2+ and BMP-6 Signaling Regulate E2F during Epidermal Keratinocyte Differentiation

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca(2+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-beta1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.

Sources of neuronal material for implantation.

The adult brain is an organ that does not have the natural ability to replace cells that have been lost through damage. Possible human interventions to rectify this situation include transplanting either developing neural tissue into the damaged host brain or transplantation of neural stem cells (cells that have the capacity to proliferate into neural cells and self-replicate) into the damaged area. Fetal or embryonic stem cells can be extracted and differentiated in vitro into the specific desired progeny (e.g. neurons). The neuronal stem cells themselves can be extracted from fetuses and multiplied in culture and then transplanted into the damaged brain. There is the possibility of dedifferentiation, in which cells of one type can be converted into a different cell type; for example, a differentiated blood cell could be de-differentiated back to its own hemopoietic stem cell and that stem cell could be converted into a neuronal stem cell which could then be differentiated into a neuron. It is probable that methods of generating large numbers of committed stem cells to treat conditions such as Alzheimer's disease will soon be increasingly common.

Liver transplantation of hepatic stem cells: potential use for treating liver diseases.

Hepatic stem cells are an alternative means for repopulating the liver after various injuries instead of liver transplantation. The first step before use is to select stem cells that will be good candidates. This review discusses the different candidates including fetal progenitor bipotential hepatic stem cells; adult hepatocytes, which can be considered as unipotential committed stem cells; and oval cells, a type of nonparenchymal pluripotential hepatic stem cell. The advantages and disadvantages of each type of cell are discussed and several other possible alternatives, such as the use of hematopoietic stem cells are analyzed.

Effect of interleukin-3, stem cell factor and granulocyte-macrophage colony-stimulating factor on committed stem cells, long-term culture initiating cells and bone marrow stroma in a one-step long-term bone marrow culture.

Long-term bone marrow culture (LTBMC) supports both differentiation and conservation of hematopoietic progenitor cells. During the culture period, the frequency and proliferative potential of early repopulating stem cells that give rise to progenitors detectable after 5 weeks in LTBMC--so-called long-term culture-initiating cells (LTC-IC)--can be investigated. The adherent stroma cell layer was modified by interleukin-3 (IL-3) + granulocyte-macrophage colony-stimulating factor (GM-CSF)+ stem-cell factor (SCF) with an increased cellularity and higher percentage of differentiated myeloid cells and a reduced percentage of stroma cells. We have studied the effects of stimulative cytokines, such as GM-CSF, SCF, and IL-3, on proliferation of committed colony-forming unit cells (CFU-C), LTC-IC, and stroma cell formation in LTBMC of unseparated bone-marrow cells. IL-3, GM-CSF, and SCF significantly stimulated the cumulative proliferation of nucleated cells and committed CFU-C. Weekly stimulation of LTBMC during the culture period did not exhaust the proliferative capacity of stem cells as seen in maintenance of LTC-IC after 5 weeks in LTBMC. In contrast, no LTC-IC were seen in limiting dilution analyses from LTBMC cultured 5 weeks without cytokine supplementation. Our data indicate that an increased proliferation of committed stem cells can be achieved by the addition of stimulatory growth factors, and maintenance of LTC-IC is possible over a period of 5 weeks. A net expansion of LTC-IC if compared with the starting bone-marrow suspension could not be obtained after a 5-week LTBMC period.

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment.

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+ cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+ cells in the bone marrow. The clonogenicity as well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38-) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+ and CD34+CD38- populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.

A biomathematical model of hematotoxicity

Pharmacodynamic models representing interactions among chemicals and cells can help to clarify how time patterns of administered dose affect risks of adverse health outcomes. This paper summarizes a model that predicts the effects of the myelotoxic and immunosuppressive drug cyclophosphamide (CP) on the hematopoietic (blood-forming) system. It consists of a set of physiological compartments representing hematopoietic progenitor cell, granulocyte-macrophage (GM)-committed stem cells, and more mature blood cells. These compartments are linked by nonlinear feedback control loops and are susceptible to first-order cell-killing kinetics from cytotoxic metabolites. The model was validated by testing its predictions against experimental and clinical data for blood cell counts following administration of CP to mice, dogs, and humans. It successfully explains apparent anomalies and patterns in previously published data, including the fact that smaller cumulative doses can cause larger hematotoxic responses. An intriguing prediction from the model is that sustained exposures to sufficiently small concentrations of myelotoxic agents may tend to provoke a protective response, increasing rather than decreasing the numbers of GM colony-forming units (CFU-GM) and early hematopoietic stem cells available to sustain hematopoiesis.