Further Proof for an Unpopular Concept: A Single Cell From Bone Marrow Can Serve as a Stem Cell for Both Hematopoiesis and Osteogenesis
Authors: Darwin J Prockop
Journal: Molecular Therapy (2013)

In science, unpopular concepts must be proven time and again. The article by Hofmann et al. in this issue of Molecular Therapy provides further proof for the unpopular idea that a single cell from bone marrow can serve as a stem cell for both hematopoiesis and osteogenesis.1Hofmann TJ Otsuru S Marino R Rasini V Veronesi E Murgia A et al.Transplanted murine long-term repopulating hematopoietic cells can differentiate to osteoblasts in the marrow stem cell niche.Mol Ther. 2013; 21: 1224-1231Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Moreover, Hofmann et al. establish that this single cell is actually an old friend: the long-term repopulating hematopoietic stem cell (LTR-HSC). These observations also suggest, but do not conclusively prove, the interesting idea that the same LTR-HSC can provide both the HSC and the microenvironmental niche required for the HSC to self-replicate and differentiate. Such a dual function can easily explain why an LTR-HSC is a long-term repopulating cell. The study provides new evidence for a refreshingly simple concept that probably should have occurred to us earlier. The story that Hofmann et al. tell has a long and tangled history. The early contributions of many scientists to this history can be readily accessed only through the titles of their publications because of the limited retrospective reach of PubMed.2Patt HM Maloney MA Bone formation and resorption as a requirement for marrow development.Proc Soc Exp Biol Med. 1972; 140: 205-207Crossref PubMed Scopus (35) Google Scholar,3Lord BI Hendry JH The distribution of haemopoietic colony-forming units in the mouse femur, and its modification by x rays.Br J Radiol. 1972; 45: 110-115Crossref PubMed Scopus (76) Google Scholar,4Gong JK Endosteal marrow: a rich source of hematopoietic stem cells.Science. 1978; 199: 1443-1445Crossref PubMed Scopus (195) Google Scholar The contributions of others have faded because of artifacts that were either not recognized at the time or as yet undiscovered. At any rate, early research on hematopoiesis clearly demonstrated a close relationship to osteogenesis. In addition, there were suggestions that both hematopoietic cells and bone cells arose from the same cell. These suggestions were based on many observations, including one from embryology that showed that bone marrow and endochondrial bone arose from the same vascular cells that invaded the initial cartilage molds of long bones. But these observations were difficult to integrate into the paradigm that quickly dominated the field, namely, that blood and bone arose from two distinct classes of stem cells. Specifically, the work of Hofmann et al. began more than 10 years ago with earlier work by the senior author of the paper, Edwin Horwitz.5Horwitz EM Prockop DJ Fitzpatrick LA Koo WW Gordon PL Neel M et al.Transplantability and therapeutic effects of bone marrow–derived mesenchymal cells in children with osteogenesis imperfecta.Nat Med. 1999; 5: 309-313Crossref PubMed Scopus (1611) Google Scholar,6Horwitz EM Gordon PL Koo WK Marx JC Neel MD McNall RY et al.Isolated allogeneic bone marrow–derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone.Proc Natl Acad Sci USA. 2002; 99: 8932-8937Crossref PubMed Scopus (1415) Google Scholar Horwitz and colleagues carried out a pioneering clinical trial with bone marrow cells in children with osteogenesis imperfecta (OI, also known as brittle bone disease). The trial was based on observations in my own laboratory with transgenic mice that developed a phenotype resembling OI because they expressed a mutated human gene for type I collagen that was discovered in a patient with OI.7Pereira RF O'Hara MD Laptev AV Halford KW Pollard MD Class R et al.Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.Proc Natl Acad Sci USA. 1998; 95: 1142-1147Crossref PubMed Scopus (489) Google Scholar The mice improved after being marrow ablated and then receiving infusions, from normal mice, of both bone marrow and genetically marked plastic-adherent cells from bone marrow that are referred to as fibroblastic colony-forming units, mesenchymal stem cells, or marrow stromal cells (MSCs).8Owen M Friedenstein AJ Stromal stem cells: marrow-derived osteogenic precursors.Ciba Found Symp. 1988; 136: 42-60PubMed Google Scholar,9Caplan AI Mesenchymal stem cells.J Orthop Res. 1991; 9: 641-650Crossref PubMed Scopus (3473) Google Scholar,10Prockop DJ Marrow stromal cells as stem cells for nonhematopoietic tissues.Science. 1997; 276: 71-74Crossref PubMed Scopus (4130) Google Scholar,11Prockop DJ Oh JY Medical therapies with adult stem/progenitor cells (MSCs): a backward journey from dramatic results in vivo to the cellular and molecular explanations.J Cell Biochem. 2012; 113: 1460-1469PubMed Google Scholar,12Keating A Mesenchymal stromal cells: new directions.Cell Stem Cell. 2012; 10 (Erratum in: Cell Stem Cell2012; 11: 136 (2012)): 709-716Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar On the basis of these observations, Horwitz and associates5Horwitz EM Prockop DJ Fitzpatrick LA Koo WW Gordon PL Neel M et al.Transplantability and therapeutic effects of bone marrow–derived mesenchymal cells in children with osteogenesis imperfecta.Nat Med. 1999; 5: 309-313Crossref PubMed Scopus (1611) Google Scholar,6Horwitz EM Gordon PL Koo WK Marx JC Neel MD McNall RY et al.Isolated allogeneic bone marrow–derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone.Proc Natl Acad Sci USA. 2002; 99: 8932-8937Crossref PubMed Scopus (1415) Google Scholar treated several children with severe OI first with a marrow transfusion from normal donors and then several years later with MSCs from the same or haplotype-matched donors. The children were not cured but showed temporary improvement in several clinical parameters. Importantly, the children exhibited no serious adverse effects. These observations, together with a subsequent observation by Le Blanc et al.,13Le Blanc K Rasmusson I Sundberg B Götherström C Hassan M Uzunel M et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet. 2004; 363: 1439-1441Abstract Full Text Full Text PDF PubMed Scopus (2283) Google Scholar who administered MSCs to a child with severe graft-versus-host disease, opened a floodgate of clinical trials with MSCs in a wide variety of diseases. More than 200 trials with MSCs or similar cells have been registered, and some have reached phase II or III (ClinicalTrials.gov). But the story took a strange and unexpected turn. Horwitz and his colleagues attempted to demonstrate engraftment of MSCs in bones of mice. To their surprise, they found that a fraction of nonadherent cells from bone marrow that contained HSCs engrafted into bone far more efficiently than plastic-adherent MSCs.14Dominici M Pritchard C Garlits JE Hofmann TJ Persons DA Horwitz EM Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation.Proc Natl Acad Sci USA. 2004; 101: 11761-11766Crossref PubMed Scopus (134) Google Scholar They published this observation more than 10 years ago. Their results were consistent with previous reports by several groups of investigators. For example, Nilsson et al.15Nilsson SK Dooner MS Weier HU Frenkel B Lian JB Stein GS et al.Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice.J Exp Med. 1999; 189: 729-734Crossref PubMed Scopus (146) Google Scholar observed that whole bone marrow could generate bone cells in nonablated mice. Olmsted-Davis et al.16Olmsted–Davis EA Gugala Z Camargo F Gannon FH Jackson K Kienstra KA et al.Primitive adult hematopoietic stem cells can function as osteoblast precursors.Proc Natl Acad Sci USA. 2003; 100: 15877-15882Crossref PubMed Scopus (154) Google Scholar found that a single adult hematopoietic cell ("side population" cell) could function as an osteoblast in mice. The publications were largely ignored by most investigators in the field, including me. Why? There were two reasons. One was that demonstrating engraftment of cells into bone is technically difficult. Sectioning of bone for microscopy requires decalcification, during which much of the cellular architecture is lost. Also, the tissue has a high degree of autofluorescence. Extracting DNA or RNA from bone is equally difficult. In addition, it is impossible to obtain quantitative data on engraftment into the whole skeleton. Therefore, the doubters in the field raised technical objections, some of which were valid. But a more compelling reason for ignoring these findings is that the results contradicted the prevailing paradigm in the field that marrow contained two distinct classes of stem cells: HSCs and MSCs. The paradigm was based on several well-documented observations, for example, that viable cultures of HSCs17Eaves CJ Cashman JD Sutherland HJ Otsuka T Humphries RK Hogge DE et al.Molecular analysis of primitive hematopoietic cell proliferation control mechanisms.Ann NY Acad Sci. 1991; 628: 298-306Crossref PubMed Scopus (44) Google Scholar required feeder layers of fibroblasts or the plastic-adherent and fibroblast-like MSCs from bone initially identified by Owen and Friedenstein.8Owen M Friedenstein AJ Stromal stem cells: marrow-derived osteogenic precursors.Ciba Found Symp. 1988; 136: 42-60PubMed Google Scholar The paradigm had tremendous appeal because it was simple and it invited research on the two kinds of stem cell. Research on MSCs was particularly appealing, because these cells could be expanded in culture far more easily than HSCs. And the research paradigm was highly productive. However, the excitement about HSCs and MSCs as separate classes of stem cells pushed aside any thought of searching for a common precursor cell, or even a cell that engrafted in bone more efficiently than MSCs. Despite the reception to their initial publications, Horwitz et al. and several other research groups18Dominici M Marino R Rasini V Spano C Paolucci P Conte P et al.Donor cell-derived osteopoiesis originates from a self-renewing stem cell with a limited regenerative contribution after transplantation.Blood. 2008; 111: 4386-4391Crossref PubMed Scopus (46) Google Scholar,19Otsuru S Gordon PL Shimono K Jethva R Marino R Phillips CL et al.Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms.Blood. 2012; 120: 1933-1941Crossref PubMed Scopus (110) Google Scholar,20Mehrotra M Rosol M Ogawa M Larue AC Amelioration of a mouse model of osteogenesis imperfecta with hematopoietic stem cell transplantation: microcomputed tomography studies.Exp Hematol. 2010; 38: 593-602Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar persevered. In their paper in this issue, Hofmann et al. provide some very convincing data based largely on experiments with bone marrow and marrow cells from transgenic mice ubiquitously expressing green fluorescent protein (GFP).1Hofmann TJ Otsuru S Marino R Rasini V Veronesi E Murgia A et al.Transplanted murine long-term repopulating hematopoietic cells can differentiate to osteoblasts in the marrow stem cell niche.Mol Ther. 2013; 21: 1224-1231Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar First they confirmed that Lin−Sca1+ and cKit+Lin−Sca1+ cells (KLS cells) from these mice generated GFP-labeled osteoblasts and osteocytes after administration to lethally irradiated mice of the same strain. Lin−Sca1− cells reconstituted short-term, but not long-term, hematopoiesis and did not give rise to osteoblasts. The authors further defined the phenotype of the osteogenic cells as CD34−KLS cells, the phenotype usually ascribed to LTR-HSCs. In a critical experiment, they demonstrated that just 300 unlabeled CD34−KLS cells competitively inhibited the engraftment into bone cells observed with unfractionated bone marrow cells from the GFP mice. Then they performed an ultimate test of stem cells: they injected a single GFP-labeled KLS cell (along with unlabeled Sca1− cells) into each of a large number of irradiated mice (n = 60). Four of the mice demonstrated significant engraftment of GFP-expressing cells into multiple tissues. Bone marrow from these mice was transplanted into secondary recipients. GFP-labeled osteoblasts and osteocytes were again found in several of the secondary recipients. The results therefore present a convincing case that cells previously shown to be LTR-HSCs are also stem cells for osteoblasts. In addition, the authors observed that the labeled osteoblasts and osteocytes in the secondary recipients were found in clusters similar to clusters of HSCs seen after marrow transplants. Therefore, the GFP-expressing osteoblasts could have contributed to a niche for the HSCs. The data presented by Hofmann et al., as well as similar observations by others, may show that we have moved too fast. We may have walked past a simple biological truth that unifies our thinking about bone marrow and stem cells in general. Embarrassingly for us all, it is a truth that was apparent from the earliest observations of stem cells in biology: stem cells do not exist in isolation. They need nurse cells or feeder cells that provide the niche to support their self-renewal and differentiation. The importance of the stem cell niche is dramatically illustrated in simple systems such as germline stem cells in DrosophilaI.21Fuller MT Spradling AC Male and female Drosophila germline stem cells: two versions of immortality.Science. 2007; 316: 402-404Crossref PubMed Scopus (332) Google Scholar The lesson was largely overlooked in research on hematopoiesis, in which it was generally assumed that HSCs were preprogrammed to differentiate via a hierarchical pattern that required prodding from only a few cytokines to define the fate of the progeny. As Orkin and Zon22Orkin SH Zon LI Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar concluded several years ago after an exhaustive review of hematopoiesis, "The 'classical' hierarchy diagram…provides a seductive, but overly simplified view." The importance of a niche for HSCs in marrow is now generally recognized, and defects in that niche may underlie many diseases.23Park D Sykes DB Scadden DT The hematopoietic stem cell niche.Front Biosci. 2012; 17: 30-39Crossref PubMed Scopus (55) Google Scholar,24Shiozawa Y Taichman RS Getting blood from bone: an emerging understanding of the role that osteoblasts play in regulating hematopoietic stem cells within their niche.Exp Hematol. 2012; 40: 685-694Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The nature of the niche is still poorly understood, but it includes MSC-like cells and cells of the osteolineage.25Méndez–Ferrer S Michurina TV Ferraro F Mazloom AR Macarthur BD Lira SA et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature. 2010; 466: 829-834Crossref PubMed Scopus (2468) Google Scholar,26Hoggatt J Scadden DT The stem cell niche: tissue physiology at a single cell level.J Clin Invest. 2012; 122: 3029-3034Crossref PubMed Scopus (31) Google Scholar If LTR-HSCs can provide both the stem cells for hematopoiesis and the cells that are required for the niche, it is no surprise that they are long-term repopulating cells. And the potential of LTR-HSCs to generate their own niches is reminiscent of the tendency of MSCs to generate their own microenvironments as they are plated at clonal densities so that colonies develop from single cells.27Prockop DJ Repair of tissues by adult stem/progenitor cells (MSCs): controversies, myths, and changing paradigms.Mol Ther. 2009; 17: 939-946Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar